Protein analysis

ABSTRACT

Disclosed, inter alia, is a method of evaluating a sample that includes a serum protein and one or more one or more compounds physically associated with the serum protein. The method can include using a peptide ligand that specifically interacts with the serum protein to analyze a complex formed by the serum protein and its associated compounds.

RELATED APPLICATIONS

[0001] This application claims benefit of priority to U.S. ProvisionalPatent Application Serial No. 60/388,642, filed Jun. 14, 2002, thecontents of which are incorporated herein by reference.

BACKGROUND

[0002] This application relates to the analysis of proteins, includingserum proteins.

[0003] Serum is the blood-derived fluid that remains after blood hasclotted. The more abundant serum proteins include serum albumin andantibodies (e.g., IgG, IgM, and the like). Other proteins that can bepresent in serum include: transferrin, α-macroglobulins, ferritin,apolipoproteins, transthyretin, protease inhibitors, retinol bindingprotein, thiostatin, α-fetoprotein, vitamin-D binding protein, andafamin (see, e.g., U.S. Pat. No. 5,767,243).

[0004] The most abundant protein component in circulating blood ofmammalian species is serum albumin, which is normally present at aconcentration of approximately 3 to 4.5 grams per 100 ml of whole blood.Serum albumin is a blood protein of approximately 70 kilo-Daltons whichprovides several important functions in the circulatory system. Forinstance, it functions as a transporter of a variety of organicmolecules found in the blood, as the main transporter of variousmetabolites such as fatty acids, hematin, and bilirubin, and, owing toits abundance, as an osmotic regulator of the circulating blood. It alsohas a broad affinity for small, negatively charged aromatic compounds.These binding functions enable serum albumin to serve as the principalcarrier of fatty acids that are otherwise insoluble in circulatingplasma. Likewise, it can sequester oxygen free radicals and toinactivate toxic lipophilic metabolites such as bilirubin. It can alsoform covalent adducts with pyridoxal phosphate cysteine, glutathione,and various metals, such as Cu(II), Ni(II) Hg(II), Ag(II), and Au(I).

[0005] Serum albumin can also bind to drugs that are present in thebody. Indeed, one indicator of the efficacy of a drug is its affinityfor serum albumin or other serum proteins. Binding to serum albumin canaffect the overall distribution, metabolism, and bioavailability of manydrugs. At least in some cases, unusually high affinity to serum albuminhas been associated with the failure of candidate drugs.

[0006] It is also known to conjugate drugs to serum albumin to extendtheir half-life and distribution. Recently, chimeric albumin moleculessuch as HSA-CD4 and HSA-Cu,Zn-superoxide dismutase have been utilized toincrease the half-life and distribution and to reduce the immunogenicityof these potential protein therapeutics.

SUMMARY

[0007] In one aspect, the invention features a method that includes:providing a sample that includes (i) a serum albumin, (ii) one or morecompounds physically associated with the serum albumin and (iii) a serumalbumin-binding agent; allowing the serum albumin-binding agent to bindto the serum albumin to form a complex; separating the complex from oneor more components of the sample; and evaluating one or more of thephysically associated compounds. The method can be used to evaluating asample. The method can further include separating one or more of thephysically associated compounds from the serum albumin, e.g., prior tothe evaluating. In one embodiment, the serum albumin is a human serumalbumin.

[0008] In an embodiment, the serum albumin-binding agent has one or moreof the following properties: is synthetic; includes a protein other thanan antibody or antibody derivative; is non-naturally occurring; is freeof an immunoglobulin variable domain; includes a peptide thatindependently binds to serum albumin.

[0009] In the latter case, the peptide can include at least oneintra-molecular disulfide bonds, e.g., one or two intra-moleculardisulfide bonds. The peptide can include a peptide described herein,e.g., DX-321, DX-321-A, DX-321-B, DX-236, DX-236-A, or DX-236B, or avariant thereof, or a peptide described in U.S. Published application2003/0069395; Ser. No. 10/094,401; Ser. No. 60/331,352; or Ser. No.60/292,975, or a variant thereof. Particular variants include:functional variants having between one and six substitutions (e.g.,between one and four, e.g., one, two, three, or four), e.g.,conservative substitutions, truncations, chemically modified forms,peptido-mimetics, and substitutions with non-naturally occurringresidues. The peptide may also include be a functional variant withbetween one and four insertions or deletions, e.g., one , two, three orfour. The peptide can be a peptide ligand that competes for binding toserum albumin with a ligand described herein, or a ligand binding anepitope that overlaps with an epitope bound by a ligand describedherein. The peptide ligand can be a ligand isolated by screening adisplay library. The peptide that independently binds to serum albumincan be less than 32, 28, 24, 20, or 16 amino acids in length, or between12 and 32, 8 and 16, or 12 and 24 amino acids in length.

[0010] In one embodiment, the serum albumin-binding agent is coupled toan insoluble support, e.g., a bead (such as a magnetic bead), a matrix(such as a chromatography matrix, agarose, or a porous material), or aplanar surface. For example, the support may include a planar surface,and the serum albumin-binding agent is immobilized to a discrete addresson the planar surface. The planar surface can also include a secondbinding agent at a second discrete address, e.g., another serumalbumin-binding agent or an agent that binds to a different serumprotein.

[0011] The serum albumin-binding agent can have a binding affinity(K_(D)) of less than 5, 4, 2, 1, 0.5, 0.1 μM, or less than 50, 10, 5, or0.5 nM and/or of greater than 0.05, 0.5, 5, or 50 nM, 0.001, 0.1, or 0.1μM, and ranges therebetween. In one embodiment, the serumalbumin-binding agent binds to serum albumin under physiologicalconditions. In one embodiment, the serum albumin-binding agent and theserum albumin preferentially dissociate at least in solutions above pH8, 8.5, or 9, e.g., between pH 8 and 11; pH 8 and 10.5; pH 8 and 10; pH8.5 and 10; or pH 8.7 and 9.5. In another embodiment, the serumalbumin-binding agent and the serum albumin preferentially dissociate atleast in solutions below pH 6, 5.5, or 6, e.g., between pH 4 and 6; pH4.6 and 6.5; pH 5 and 6.5; or pH 4.7 and 6.0.

[0012] In one embodiment, the serum albumin-binding agent is less than7, 5, 3, or 2 kDa molecular weight or between 1.5 and 7 or 2 and 6 kDamolecular weight.

[0013] The serum albumin-binding agent may bind to serum albumin from aplurality of species, e.g., a plurality of mammalian species, e.g.,human and mouse. In another embodiment, the serum albumin-binding agentbinds to human serum albumin but not murine serum albumin nor bovineserum albumin.

[0014] In one embodiment, at least one of the evaluated physicallyassociated compounds is non-covalently associated with the serumalbumin. Such compounds may be directly or indirectly physicallyassociated with the serum albumin. An indirect interaction may bebridged by one or more compounds, at least one of which is directlyassociated with the serum albumin. In another embodiment, at least oneof the evaluated physically associated compounds is covalentlyassociated with the serum albumin. In some embodiments, at least one ofthe evaluated physically associated compounds is covalently associatedand at least another is non-covalently associated.

[0015] The method can include further including separating the at leastone non-covalently associated compounds from the serum albumin, e.g.,prior to the evaluating. The separating from the serum albumin caninclude covalently attaching the serum albumin to an insoluble support,e.g., a matrix, a particle, or a surface. For example, the covalentattachment can be to a free cysteine of the serum albumin. The covalentattachment can be formed using a thiol reactive group, e.g., a halogenderivative (such as iodoacetamide), a maleimide, or a thiol exchangereagent (e.g., a pyridyl disulfide).

[0016] The separating can include denaturing the serum albumin, e.g.,using a chaotrope, an organic solvent, high or low pH, or heat. Inanother embodiment, where at least one of the evaluated covalentlyassociated compounds is protease resistant (e.g., includes anon-proteinaceous component), the separating can include degrading theserum albumin, e.g., using a protease.

[0017] The evaluating can include one or more of: gel electrophoresis,mass spectroscopy, chromatography, protein sequencing, detecting a label(e.g., a radioactive, fluorescent, enzymatic, or chemical label),detecting a given compound using an affinity reagent specific for thegiven compound, or another method described herein. The affinity reagentmay be an antibody. For example, the detecting can include performing animmuno-blot or an immuno-precipitation. Information from the evaluatingcan be recorded on a machine-readable medium, transmitted across acomputer network, or stored in a database.

[0018] The subject of the evaluating can include a proteinaceous or anon-proteinaceous chemical compound. For example, the subject caninclude a peptide, a polypeptide, a protein complex, or a drug. In oneembodiment, the compound is other than one or more of the compounds inTable 1 or Table 2, e.g., a compound other than a fatty acid, hematin,bilirubin, or an exogenous compound.

[0019] In one embodiment, the evaluating includes eluting an associatedcompound from the serum albumin by competition using a syntheticaffinity ligand specific for an epitope of the serum albumin or anatural compound (e.g., a fatty acid, hematin, and bilirubin) that bindsto the serum albumin. The natural compound can include a negativelycharged aromatic group having a molecular weight of less than 500Daltons.

[0020] In one embodiment, the serum albumin is an artificial mutant of anaturally-occurring serum albumin. For example, the serum albumin can befused to a heterologous polypeptide or covalently coupled to atherapeutic agent (e.g., a cytotoxic drug).

[0021] The method can further include digitally recording informationthat (i) indicates the presences or absence of a given compound amongthe evaluated one or more physically associated compounds, or (ii)describes the one or more physically associated compounds.

[0022] In one embodiment, the sample is obtained from a subject, e.g., ahuman, e.g., a patient. The sample may include blood or serum. Inanother example, the sample is obtained from a biopsy, e.g., obtainedfrom a tumor, a region adjacent to a tumor, or a lymph node. The subjectmay be treated with a therapeutic composition prior to obtaining thesample.

[0023] In one embodiment, one or more of the evaluated physicallyassociated compounds is an endogenous compound. In another embodiment,one or more of the evaluated physically associated compounds is acomponent of the therapeutic composition.

[0024] In one embodiment, the method further includes providing a secondsample, and evaluating one or more of the physically associatedcompounds in the second sample. The method can further include comparingresults of evaluating the one or more of the physically associatedcompounds for the first sample to the second sample.

[0025] In one embodiment, the first and second samples are obtained froma first and a second subject, respectively. In one example, the firstsubject and second subject are respectively treated with an agent anduntreated with the agent, e.g., a small molecule. The agent may beadministered parenterally. In another example, the first subject andsecond subject are subjected to different environmental conditions. Instill another example, the first subject is a reference subject and thesecond subject is an experimental subject. In another example, the firstsubject is a reference subject and the second subject is an affectedand/or diseased subject. In still another example, the first and secondsamples are obtained from the same subject, e.g., at different times,e.g., at different times during a treatment.

[0026] The results can be recorded in a machine or on machine-readablemedia. For example, the results are stored in a computer database.

[0027] In one embodiment, the results for the first and second samplesare compared to a reference sample. In one embodiment, results for thefirst and second samples are compared to a database that includesrecords for samples, each sample record being associated withinformation about the sample (e.g., origin, disease, environmentalcondition, physiological condition, and so forth).

[0028] In another aspect, the invention features a method that includesproviding a sample that includes a serum albumin, one or more compoundsassociated with the serum albumin, and a component that does notassociate with the serum albumin; contacting the sample to an affinityligand specific for the serum albumin; and separating the un-associatedcomponent from a composition that includes the serum albumin and one ormore of the associated compounds, thereby providing a serumalbumin-associated compound. The method can be used to provide a serumalbumin-associated compound. The method can include other featuresdescribed herein. The invention also provides a composition prepared bythe above method or a method described herein.

[0029] The method can further include separating the associated compoundfrom the serum albumin to provide a serum-albumin free preparation. Theinvention also features a serum-albumin free preparation preparedaccording to the above method or another method described herein.

[0030] In another aspect, the invention features a method that includesproviding (e.g., receiving or obtaining) a first and second sample thateach includes a serum protein (e.g., a serum albumin, a solubleimmunoglobulin, or other serum protein); evaluating each sample forassociated compound(s), if present, e.g., according to a methoddescribed herein; and comparing results of the evaluating for the firstand second samples. The method can further include, prior to theevaluating, isolating the serum protein and compounds associated withthe serum protein from each sample. The separating can includecovalently attaching the serum protein to an insoluble matrix. The serumprotein can be an abundant serum protein, e.g., a serum protein that isforms at least 0.01, 0.05, or 0.1% of the blood serum.

[0031] In one embodiment, the first and second samples are obtained froma first and a second subject, respectively. In one example, the firstsubject and second subject are respectively treated with an agent anduntreated with the agent, e.g., a small molecule. The agent may beadministered parenterally. In another example, the first subject andsecond subject are subjected to different environmental conditions. Instill another example, the first subject is a reference subject and thesecond subject is an experimental subject. In another example, the firstsubject is a reference subject and the second subject is an affectedand/or diseased subject.

[0032] The results can be recorded in a machine or on machine-readablemedia. For example, the results are stored in a computer database.

[0033] In one embodiment, the results for the first and second samplesare compared to a reference sample. In one embodiment, results for thefirst and second samples are compared to a database that includesrecords for samples, each sample record being associated withinformation about the sample (e.g., origin, disease, environmentalcondition, physiological condition, and so forth).

[0034] The method can also include other features described herein.

[0035] In another aspect, the invention features a method that includes:providing a sample that includes (i) a soluble immunoglobulin proteinthat includes at least one immunoglobulin domain (ii) one or morecompounds physically associated with the soluble immunoglobulin proteinand (iii) a peptide immunoglobulin-binding agent; allowing theimmunoglobulin-binding agent to bind to the soluble immunoglobulinprotein to form a complex that includes one or more compounds physicallyassociated with the soluble immunoglobulin protein; separating thecomplex from one or more components of the sample; and evaluating one ormore of the physically associated compounds. The method can be used toevaluate a sample.

[0036] In one embodiment, the soluble immunoglobulin protein is anaturally-occurring protein, e.g., IgG, IgM, IgA, IgE, or IgD. Inanother embodiment, the soluble immunoglobulin protein is a Fab orsingle-chain antibody. Such protein may include at least one syntheticcomplementarity determining region (CDR).

[0037] In one embodiment, the one or more physically associatedcompounds includes an antigen of a pathogen.

[0038] The sample can be obtained from a subject having an infection,immunological disorder (e.g., an auto-immune disorder), or a geneticdisorder. The subject may also be a normal subject.

[0039] In an embodiment, the immunoglobulin-binding agent has one ormore of the following properties: is synthetic; includes a protein otherthan an antibody or antibody derivative; is non-naturally occurring; isfree of an immunoglobulin variable domain; includes a peptide thatindependently binds to immunoglobulin. The immunoglobulin-binding agentcan bind to the Fc region, to a constant domain (e.g., CH1, CH2, CH3,CH4, or CL), or to a framework region of a variable domain. In aprincipal embodiment, the immunoglobulin binding agent does not bind tothe antigen-binding site of an immunoglobulin.

[0040] The peptide can include one or more intra-molecular disulfidebonds, e.g., one or two intra-molecular disulfide bonds. In the case ofan immunoglobulin binding agent that binds to an Fc region, the peptidecan include a peptide described herein, e.g., DX249, DX249-A, DX249-B,DX253, DX253-A, DX253-B, DX398, DX398-A, DX398-B or a variant thereof,or a compound described in Ser. No. 10/125,869, filed Apr. 18, 2002, ora variant thereof. Exemplary variants include: functional variantshaving between one and six substitutions, e.g., conservativesubstitutions, truncations, chemically modified forms, peptido-mimetics,and substitutions with non-naturally occurring residues. The peptide canbe a peptide ligand that competes for binding to an immunoglobulin witha ligand described herein, or a ligand binding an epitope that overlapswith an epitope bound by a ligand described herein. The peptide ligandcan be a ligand isolated by screening a display library. The peptidethat independently binds to an immunoglobulin can be less than 32, 28,24, 20, or 16 amino acids in length, or between 12 and 32, 8 and 16, or12 and 24 amino acids in length.

[0041] In one embodiment, the immunoglobulin-binding agent is coupled toan insoluble support, e.g., a bead (such as a magnetic bead), a matrix(such as a chromatography matrix), or a planar surface. For example, thesupport may include a planar surface, and the immunoglobulin-bindingagent is immobilized to a discrete address on the planar surface. Theplanar surface can also include a second binding agent at a seconddiscrete address, e.g., another immunoglobulin-binding agent or an agentthat binds to a different serum protein.

[0042] The immunoglobulin-binding agent can have a binding affinity(K_(D)) of less than 5, 4, 2, 1, 0.5, or 0.1 μM and/or of greater than0.001, 0.1, or 0.1 μM, and ranges therebetween. In one embodiment, theimmunoglobulin-binding agent binds to immunoglobulin under physiologicalconditions.

[0043] In one embodiment, the immunoglobulin-binding agent is less than7, 5, 3, or 2 kDa molecular weight or between 1.5 and 7 or 2 and 6 kDamolecular weight.

[0044] The immunoglobulin-binding agent may bind to immunoglobulins froma plurality of species, e.g., a plurality of mammalian species, e.g.,human and mouse. In another embodiment, the immunoglobulin-binding agentbinds to a human immunoglobulin but not a murine immunoglobulin.

[0045] In one embodiment, at least one of the evaluated physicallyassociated compounds is non-covalently associated with theimmunoglobulin. Such compounds may be directly or indirectly physicallyassociated with the immunoglobulin. An indirect interaction may bebridged by one or more compounds, at least one of which is directlyassociated with the immunoglobulin. In one embodiment, an associatedcompound is an antigen recognized by the immunoglobulin. For example, anantigen that is a component of a pathogen, e.g., a virus or bacterium,e.g., a replicable virus or live bacterium.

[0046] The method can include further including separating the at leastone non-covalently associated compounds from the immunoglobulin, e.g.,prior to the evaluating. The separating from the immunoglobulin caninclude covalently attaching the immunoglobulin to an insoluble support,e.g., a matrix, a particle, or a surface.

[0047] The separating can include denaturing the immunoglobulin, e.g.,using a chaotrope, an organic solvent, high or low pH, or heat. Inanother embodiment, wherein at least one of the evaluated associatedcompounds is protease resistant (e.g., includes a non-proteinaceouscomponent), the separating can include degrading the immuno-globulin.

[0048] The evaluating can include one or more of: gel electrophoresis,mass spectroscopy, chromatography, protein sequencing, detecting a label(e.g., a radioactive, fluorescent, enzymatic, or chemical label),detecting a given compound using an affinity reagent specific for thegiven compound, or another method described herein. The affinity reagentmay be an antibody. For example, the detecting can include performing animmuno-blot or an immuno-precipitation.

[0049] The evaluating can include culturing a pathogen (e.g., virus orbacterium) that is associated with the immunoglobulin.

[0050] The subject of the evaluating can include a proteinaceous or anon-proteinaceous chemical compound. For example, the subject caninclude a peptide, a polypeptide, a protein complex, or a drug. In oneembodiment, the compound is other than an antigen, e.g., the compound isassociated with the immunoglobulin by interactions outside the CDRregion.

[0051] In one embodiment, the evaluating includes eluting an associatedcompound from the immunoglobulin by competition using a syntheticaffinity ligand specific for an epitope of the immunoglobulin or anantigen. The natural compound can include a negatively charged aromaticgroup having a molecular weight of less than 500 Daltons.

[0052] In one embodiment, the immunoglobulin is an artificial variant ofa naturally-occurring immunoglobulin. For example, the immunoglobulincan be fused to a heterologous polypeptide or covalently coupled to atherapeutic agent (e.g., a cytotoxic drug).

[0053] The method can further include digitally recording informationthat (i) indicates the presences or absence of a given compound amongthe evaluated one or more physically associated compounds, or (ii)describes the one or more physically associated compounds.

[0054] In one embodiment, the method further includes providing a secondsample, and evaluating one or more of the physically associatedcompounds in the second sample. The method can further include comparingresults of evaluating the one or more of the physically associatedcompounds for the first sample to the second sample.

[0055] In one embodiment, the sample is obtained from a subject, e.g., ahuman, e.g., a patient. The sample may include blood or serum. Inanother example, the sample is obtained from a biopsy, e.g., obtainedfrom a tumor, a region adjacent to a tumor, or a lymph node. The subjectmay be treated with a therapeutic composition prior to obtaining thesample.

[0056] In one embodiment, one or more of the evaluated physicallyassociated compounds is an endogenous compound. In another embodiment,one or more of the evaluated physically associated compounds is acomponent of the therapeutic composition.

[0057] In still another aspect, the invention features a method thatincludes: providing a complex including a serum albumin and anassociated compound; evaluating binding of a non-antibody ligand (e.g.,a peptide ligand described herein) to the complex, wherein thenon-antibody ligand binds to serum albumin, e.g., with an affinity ofless than 5, 3, 2, 1, 0.5, or 0.1 μM and binding of the non-antibodyligand to the complex indicates that the associated compound does notbind an epitope that overlaps the epitope bound by the non-antibodyligand. The method can be used to map a physical interaction betweenserum albumin and an associated compound. The method can also be varied,e.g., by first binding the ligand and then binding the associatedcompound.

[0058] The method can further include: evaluating binding of a ligand tothe complex, wherein the second ligand binds to serum albumin, e.g.,with an affinity of less than 5, 3, 2, 1, 0.5, or 0.1 μM. For example,the second ligand is other than an antibody, e.g., a peptide ligand. Inone embodiment, one of the first and second non-antibody ligand binds isprevented from binding to the complex. For example, the associatedcompound sterically hinders binding of the non-antibody ligand to serumalbumin or occludes the binding site of the non-antibody ligand forserum albumin. The ligand can be a ligand described herein. The methodcan also be varied, e.g., by first binding the ligands and then bindingthe associated compound.

[0059] In a related aspect, the invention features a method thatincludes: providing a complex including a serum albumin and anon-antibody ligand, and evaluating binding of a given compound to thecomplex. The given compound can be a compound known to bind to serumalbumin or a compound isolated from a sample in association with serumalbumin, e.g., by a method described herein. The method can be used tomap a physical interaction between serum albumin and a given compound.

[0060] The method can be repeated for a second ligand. In oneembodiment, the second ligand does not include an antigen bindingimmunoglobulin domain.

[0061] The method can be repeated for a second given compound.

[0062] The method can include other features described herein.

[0063] In another aspect, the invention features a database, including(i) data describing compounds associated with a serum protein in asample; and (ii) data indicating information about the sample, whereininstances of (i) are linked to instances of (ii). The data can beobtained from results of a method described herein.

[0064] In another aspect, the invention features a method (e.g., amachine-based method) that includes: receiving information aboutcompounds associated with a serum protein in a given sample; comparingthe information to a database that includes information about compoundsassociated with the serum protein in a plurality of reference samples tolocate information about a compound or a sample indicated by thereceived information; and providing the located information or areference to the located information to a user. The method can includeother features described herein.

[0065] In still another aspect, the invention features amachine-readable medium having encoded thereon information representinga separation process that separates compounds in a composition describedherein and/or information representing a characteristic (e.g., physicalcharacteristic, chemical structure, and so forth) of a compoundassociated with a serum protein (e.g., a serum albumin or animmunoglobulin).

[0066] The invention also features an image of a two-dimensional gelthat separates a composition described herein. Also featured is adatabase including a plurality of images, each image corresponding to atwo-dimensional gel separation of a composition described herein.

[0067] Also featured is a machine-readable medium having encoded thereoninformation representing characteristics of a plurality of compoundsdetectable in a composition described herein. Exemplary characteristicsinclude molecular weight, isoelectric point, sequence, chemicalcomposition, abundance, proteolytic fragment profile, and so forth.

[0068] In another aspect, the invention features a method that includesa sample that includes (i) a serum protein, (ii) one or more compoundphysically associated with the serum protein and (iii) a serumprotein-binding agent; allowing the serum protein-binding agent to bindto the serum protein to form a complex; separating said complex from oneor more components of the sample; and evaluating one or more of thephysically associated compounds. Examples of serum proteins includeserum albumin, antibodies (e.g., IgG, IgM, and so forth), transferrin,α-macroglobulins, ferritin, apolipoproteins, transthyretin, proteaseinhibitors, retinol binding protein, thiostatin, α-fetoprotein,vitamin-D binding protein, and afamin. The method can include otherfeatures, e.g., as described above and elsewhere herein.

[0069] In one embodiment, the method includes obtaining the sample froma subject. For example, the subject may have a metabolic disorder, andthe serum protein is a non-albumin carrier protein for one or moremetabolites.

[0070] In another related aspect, the invention features a method thatincludes: providing a sample that comprises a serum albumin having oneor more compounds physically associated with the serum albumin;isolating the serum albumin and one or more compounds physicallyassociated with the serum albumin from the sample using an affinityreagent that binds the serum albumin; and detecting the one or morephysically associated compounds. The method can be used for detecting aserum albumin-associated compound.

[0071] In one embodiment, the affinity reagent includes a proteinaceousligand that is does not have an antigen-binding immunoglobulin domain.For example, the proteinaceous ligand is a peptide ligand that bindsserum albumin, e.g., with an affinity of less than 5, 4, 2, 1, 0.5, or0.1 μM. The affinity reagent can include one or more peptide ligandsdescribed herein, e.g., DX-236 and DX-321. In one embodiment, theaffinity reagent includes two ligands that bind different epitopes. Themethod can include other features, e.g., as described above andelsewhere herein.

[0072] In still other aspects, the invention features a method thatincludes administering a composition that comprises a compound to asubject and determining association of the compound with a serum proteinfrom the subject. The determining can include covalently ornon-covalently binding the serum protein (e.g., a serum albumin) fromthe subject to an affinity reagent, e.g., a ligand described herein. Themethod can include one or more other features described herein.

[0073] In another aspect, the invention features a method that includescontacting a serum albumin to a given compound; binding the serumalbumin to an affinity reagent described herein; and determiningassociation of the given compound to the serum albumin. The method caninclude one or more other features described herein.

[0074] The method can be used for discovering associations between serumproteins and natural compounds, and, similarly, associations betweenserum proteins and non-natural compounds, such as pharmaceuticals. Themethod can also be used to characterize a subject (e.g., a human patientor an animal) by the profile of compounds associated with a given serumprotein (e.g., serum albumin).

[0075] In some embodiments, peptide ligands are used as affinityreagents to bind a serum protein. Peptide ligands offer severaladvantages. For example, the mass per binding site is low, e.g., suchlow molecular weight peptide domains can show higher bind activity pergram than larger proteins such as antibodies. The possibility ofnon-specific binding is reduced because there is only a small surfaceavailable. Peptides can be engineered to have unique tethering sitessuch as N-terminal Ser or Thr residues or terminal single or multiplelysine segments, e.g., by chemical synthetic methods. (N-terminal Ser orThr can be specifically oxidized to aldehydes that can be joined toother molecules with high specificity.) Further, as used in someembodiments, a constrained peptide structure is likely to retain itsfunctionality in a variety of contexts.

[0076] The invention also features isolated preparations of anendogenous compound associated with a serum albumin. The preparationscan be isolated by a method described herein. The preparation caninclude a single species that also be at least 50, 60, 70, 80, 90, or95% pure (weight/volume). The species can have an isoelectric point andmolecular weight according to a species isolated in FIG. 1.

[0077] An example of a non-naturally occurring, serum albumin-bindingagent is a polypeptide comprising the amino acid sequence of:

[0078] Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Cys (SEQ ID NO: 1),

[0079] wherein Xaa₁ is Asp, Asn, Ser, Thr, or Trp; Xaa₂ is Asn, Gln,His, lie, Leu, or Lys; Xaa₃ is Ala, Asp, Phe, Trp, or Tyr; and Xaa₄ isAsp, Gly, Leu, Phe, Ser, or Thr.

[0080] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0081] Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Cys-Xaa₈-Xaa₉-Xaa₁₀ (SEQID NO: 2),

[0082] wherein Xaa₁ is Asn, His, Leu, Phe, Trp, or Val; Xaa₂ is Ala,Glu, His, Lys, Trp, or Val; Xaa₃ is Asp, Gly, Ile, His, Ser, Trp, orVal; Xaa₄ is Asp, Asn, Ser, Thr, or Trp; Xaa₅ is Asn, Gln, His, Ile,Leu, or Lys; Xaa₆ is Ala, Asp, Phe, Trp, or Tyr; Xaa₇ is Asp, Gly, Leu,Phe, Ser, or Thr; Xaa₈ is Glu, Ile, Leu, Met, Ser, or Val; Xaa₉ is Asn,Asp, Gln, Gly, Met, Ser, or Trp; and Xaa₁₀ is Ala, Asn, Asp, Pro, Tyr,or Val.

[0083] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0084]Ala-Glu-Gly-Thr-Gly-Ser-Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Cys-Xaa₈-Xaa₉-Xaa₁₀-Ala-Pro-Glu(SEQ ID NO: 3),

[0085] wherein Xaa₁ is Asn, His, Leu, Phe, Trp, or Val; Xaa₂ is Ala,Glu, His, Lys, Trp, or Val; Xaa₃ is Asp, Gly, Ile, His, Ser, Trp, orVal; Xaa₄ is Asp, Asn, Ser, Thr, or Trp; Xaa₅ is Asn, Gln, His, Ile,Leu, or Lys; Xaa₆ is Ala, Asp, Phe, Trp, or Tyr; Xaa₇ is Asp, Gly, Leu,Phe, Ser, or Thr; Xaa₈ is Glu, Ile, Leu, Met, Ser, or Val; Xaa₉ is Asn,Asp, Gln, Gly, Met, Ser, or Trp; and Xaa₁₀ is Ala, Asn, Asp, Pro, Tyr,or Val.

[0086] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0087] Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Cys (SEQ ID NO: 431)

[0088] wherein Xaa₁ is Ala, Leu, His, Met, Phe, Ser, or Thr; Xaa₂ isIle, Phe, Pro, Ser, Trp, or Tyr; Xaa₃ is Asp, Gln, Glu, Lys, Pro, Trp,or Tyr; Xaa₄ is Asp, Gln, Gly, Leu, Pro, or Trp; Xaa₅ is Asp, Ile, Leu,Lys, Met, Pro, Trp, or Tyr; and Xaa₆ is Gln, Gly, Ile, Phe, Thr, Trp, orVal.

[0089] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0090]Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaas-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys-Xaa₁₀-Xaa₁₁-Xaa₁₂(SEQ ID NO: 99), wherein Xaa₁ is Ala, Gln, Leu, Lys, Phe, Trp, orTyr;Xaa₂ is Asn, Gln, Glu, Ile, Thr, or Trp; Xaa₃ is Asn, Gly, Phe, Thr,Trp, or Tyr; Xaa₄ is Ala, Leu, His, Met, Phe, Ser, or Thr; Xaa₅ is Ile,Phe, Pro, Ser, Trp, or Tyr; Xaa₆ is Asp, Gln, Glu, Lys, Pro, Trp, orTyr; Xaa₇ is Asp, Gln, Gly, Leu, Pro, or Trp; Xaa₈ is Asp, Ile, Leu,Lys, Met, Pro, Trp, or Tyr; Xaa₉ is Gln, Gly, Ile, Phe, Thr, Trp, orVal; Xaa₁₀ is Asp, Glu, Gly, Leu, Lys, Pro, or Ser; Xaa₁₁ is Glu, His,Ile, Leu, Lys, Ser, Trp, or Val; and Xaa₁₂ is Ala, Asn, His, Ile, Met,Phe, Pro, or Ser.

[0091] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0092]Ala-Gly-Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys-Xaa₁₀-Xaa₁₁-Xaa₁₂-Gly-Thr(SEQ ID NO: 100),

[0093] wherein Xaa₁ is Ala, Gln, Leu, Lys, Phe, Trp, or Tyr; Xaa₂ isAsn, Gln, Glu, Ile, Thr, or Trp; Xaa₃ is Asn, Gly, Phe, Thr, Trp, orTyr; Xaa₄ is Ala, Leu, His, Met, Phe, Ser, or Thr; Xaa₅ is Ile, Phe,Pro, Ser, Trp, or Tyr; Xaa₆ is Asp, Gln, Glu, Lys, Pro, Trp, or Tyr;Xaa₇ is Asp, Gln, Gly, Leu, Pro, or Trp; Xaa₈ is Asp, Ile, Leu, Lys,Met, Pro, Trp, or Tyr; Xaa₉ is Gln, Gly, Ile, Phe, Thr, Trp, or Val;Xaa₁₀ is Asp, Glu, Gly, Leu, Lys, Pro, or Ser; Xaa₁₁ is Glu, His, Ile,Leu, Lys, Ser, Trp, or Val; and Xaa₁₂ is Ala, Asn, His, Ile, Met, Phe,Pro, or Ser.

[0094] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0095] Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Cys (SEQ ID NO: 101),

[0096] wherein Xaa₁ is Gln, Glu, Phe, or Met; Xaa₂ is Asp, Pro, or Thr;Xaa₃ is Ile, Ser, or Trp; Xaa₄ is His, Met, Phe or Pro; Xaa₅ is Asn,Leu, or Thr; Xaa₆ is Arg, Asn, His, or Thr; Xaa₇ is Arg, Met, Phe, orTyr; and Xaa₈ is Asp, Gly, Phe, or Trp.

[0097] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0098]Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Cys-Xaa₁₂-Xaa₁₃-Xaa₁₄(SEQ ID NO: 102),

[0099] wherein Xaa₁ is Arg, Phe, or Tyr; Xaa₂ is Arg, Leu, Ser, or Trp;Xaa₃ is Asn, Asp, Phe, or Tyr; Xaa₄ is Gln, Glu, Phe, or Met; Xaa₅ isAsp, Pro, or Thr; Xaa₆ is Ile, Ser, or Trp; Xaa₇ is His, Met, Phe orPro; Xaa₈ is Asn, Leu, or Thr; Xaa₉ is Arg, Asn, His, or Thr; Xaa₁₀ isArg, Met, Phe, or Tyr; Xaa₁₁ is Asp, Gly, Phe, or Trp; Xaa₁₂ is Ala,Asn, or Asp; Xaa₁₃ is Arg, Phe, Pro, or Tyr; and Xaa₁₄ is Arg, His, Phe,or Ser.

[0100] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0101]Gly-Ser-Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Cys-Xaa₁₂-Xaa₁₃-Xaa₁₄-Ala-Pro(SEQ ID NO: 103),

[0102] wherein Xaa₁ is Arg, Phe, or Tyr; Xaa₂ is Arg, Leu, Ser, or Trp;Xaa₃ is Asn, Asp, Phe, or Tyr; Xaa₄ is Gln, Glu, Phe, or Met; Xaa₅ isAsp, Pro, or Thr; Xaa₆ is Ile, Ser, or Trp; Xaa₇ is His, Met, Phe orPro; Xaa₈ is Asn, Leu, or Thr; Xaa₉ is Arg, Asn, His, or Thr; Xaa₁₀ isArg, Met, Phe, or Tyr; Xaa₁₁ is Asp, Gly, Phe, or Trp; Xaa₁₂ is Ala,Asn, or Asp; Xaa₁₃ is Arg, Phe, Pro, or Tyr; and Xaa₁₄ is Arg, His, Phe,or Ser.

[0103] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0104] Cys-Xaa₁-Xaa₂-Xaa₃-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Cys (SEQID NO: 4),

[0105] wherein Xaa₁ is Ala, Asn, Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe,Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₂ is Ala, Arg, Asp, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Xaa₃ is Ala, Arg,Asp, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val;Xaa₄ is Ala, Arg, Asn, Asp, Ile, Leu, Phe, Pro, Ser, Trp, or Tyr; Xaa₅is Ala, Asp, Glu, Gly, Ile, Met, Phe, Pro, Thr, Trp, or Tyr; Xaa₆ isAla, Arg, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr, Trp, orTyr; Xaa₇ is Ala, Arg, Asp, Glu, Gly, His, Met, Phe, Pro, Ser, Thr, orTrp; Xaa₈ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu, Met, Phe, Pro, Ser,Thr, Trp, or Val; Xaa₉ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₁₀ is Ala, Asp, Gln, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val.

[0106] Another example of a non-naturally occurring, serumalbumin-binding agent is a polypeptide comprising the amino acidsequence of:

[0107]Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Cys-Xaa₁₄-Xaa₁₅-Xaa₁₆(SEQ ID NO: 5),

[0108] wherein Xaa₁ is Ala, Arg, Asp, Asn, Gly, His, Leu, Phe, Pro, Ser,Trp, Tyr; Xaa₂ is Ala, Arg, Asp, Asn, Gly, His, Phe, Pro, Ser, or Trp;Xaa₃ is Ala, Asn, Asp, Gln, Glu, Gly, His, Leu, Met, Phe, Ser, Thr, Trp,Tyr, or Val; Xaa₄ is Ala, Asn, Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe,Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₅ is Ala, Arg, Asp, Glu, Gly, His,Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val; Xaa₆ is Ala, Arg,Asp, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr, Trp, Tyr, or Val;Xaa₇ is Ala, Arg, Asn, Asp, Ile, Leu, Phe, Pro, Ser, Trp, or Tyr; Xaa₈is Ala, Asp, Glu, Gly, Ile, Met, Phe, Pro, Thr, Trp, or Tyr; Xaa₉ isAla, Arg, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Phe, Ser, Thr, Trp, orTyr; Xaa₁₀ is Ala, Arg, Asp, Glu, Gly, His, Met, Phe, Pro, Ser, Thr, orTrp; Xaa₁₁ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu, Met, Phe, Pro,Ser, Thr, Trp, or Val; Xaa₁₂ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu,Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₁₃ is Ala, Asp, Gln,Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;Xaa₁₄ is Ala, Arg, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Pro,Ser, Thr, Trp, or Tyr; Xaa₁₅ is Ala, Arg, Asn, Asp, Gly, His, Leu, Phe,Pro, Ser, Trp, or Tyr; and Xaa₁₆ is Ala, Asn, Asp, Gly, His, Leu, Phe,Pro, Ser, Trp, or Tyr.

[0109] Further examples of serum albumin-binding ligands that have thestructure of SEQ ID NO: 5, above, include polypeptides comprising theamino acid sequence (A) or (B):

[0110] (A)Xaa₁-Arg-Xaa₂-Cys-Xaa₃-Thr-Xaa₄-Xaa₅-Pro-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Cys-Xaa₁₁-Xaa₁₂-Xaa₁₃(SEQ ID NO: 425),

[0111] wherein Xaa₁ is Asn, Leu, or Phe, preferably Leu; Xaa₂ is Ala,Asn, Asp, Gln, Glu, Gly, His, Leu, Met, Phe, Ser, Thr, Trp, Tyr, or Val;Xaa₃ is Ala, Asn, Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Pro, Ser, Thr,Trp, Tyr, or Val; Xaa₄ is Ala, Arg, Asp, Gln, Glu, Gly, Ile, Leu, Lys,Met, Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₅ is Phe, Trp, or Tyr,preferably Trp; Xaa₆ is His or Phe, preferably Phe; Xaa₇ is Asp, Glu, orThr; Xaa₈ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu, Met, Phe, Pro, Ser,Thr, Trp, or Val; Xaa₉ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₁₀ is Ala, Asp, Gln, Glu,Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₁₁ isPro or Ser; Xaa₁₂ is Asn or Pro; and Xaa₁₃ is Asn or Pro; or

[0112] (B)Xaa₁-Xaa₂-Xaa₃-Cys-Ile-Thr-Xaa₄-Pro-Phe-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Cys-Xaa₁₀-Asn-Xaa₁₁(SEQ ID NO: 426),

[0113] wherein Xaa₁ is Ala, Arg, Asp, Asn, Gly, His, Leu, Phe, Pro, Ser,Trp, Tyr; Xaa₂ is Ala, Arg, Asp, Asn, Gly, His, Phe, Pro, Ser, or Trp;Xaa₃ is Glu, Leu, or Met, preferably Met; Xaa₄ is Trp or Tyr, preferablyTrp; Xaa₅ is Gln, Glu, or Lys; Xaa₆ is Ala, Arg, Asp, Glu, Gly, His,Met, Phe, Pro, Ser, Thr, or Trp; Xaa₇ is Met, Pro, or Ser, preferablyPro; Xaa₈ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu, Lys, Met, Phe, Pro,Ser, Thr, Trp, Tyr, or Val; Xaa₉ is His or Pro, preferably Pro; Xaa₁₀ isAla, Arg, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Pro, Ser, Thr,Trp, or Tyr; and Xaa₁₁ is Ala, Asn, Asp, Gly, His, Leu, Phe, Pro, Ser,Trp, or Tyr.

[0114] Still other non-naturally occurring, serum albumin-bindingligands include a polypeptide comprising the amino acid sequence of:

[0115]Ala-Glu-Gly-Thr-Gly-Xaa₀-Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Cys-Xaa₁₄-Xaa₁₅-Xaa₁₆-Xaa₁₇-Pro-Glu(SEQ ID NO: 6),

[0116] wherein Xaa₀ is Ala or Asp; Xaa₁ is Ala, Arg, Asp, Asn, Gly, His,Leu, Phe, Pro, Ser, Trp, Tyr; Xaa₂ is Ala, Arg, Asp, Asn, Gly, His, Phe,Pro, Ser, or Trp; Xaa₃ is Ala, Asn, Asp, Gln, Glu, Gly, His, Leu, Met,Phe, Ser, Thr, Trp, Tyr, or Val; Xaa₄ is Ala, Asn, Asp, Gln, Glu, Gly,Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₅ is Ala, Arg,Asp, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, or Val;Xaa₆ is Ala, Arg, Asp, Gln, Glu, Gly, Ile, Leu, Lys, Met, Pro, Ser, Thr,Trp, Tyr, or Val; Xaa₇ is Ala, Arg, Asn, Asp, Ile, Leu, Phe, Pro, Ser,Trp, or Tyr; Xaa₈ is Ala, Asp, Glu, Gly, Ile, Met, Phe, Pro, Thr, Trp,or Tyr; Xaa₉ is Ala, Arg, Asn, Asp, Gln, Glu, His, Ile, Leu, Lys, Phe,Ser, Thr, Trp, or Tyr; Xaa₁₀ is Ala, Arg, Asp, Glu, Gly, His, Met, Phe,Pro, Ser, Thr, or Trp; Xaa₁₁ is Ala, Arg, Asp, Gln, Glu, His, Ile, Leu,Met, Phe, Pro, Ser, Thr, Trp, or Val; Xaa₁₂ is Ala, Arg, Asp, Gln, Glu,His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; Xaa₁₃ isAla, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Phe, Pro, Ser, Thr, Trp,Tyr, or Val; Xaa₁₄ is Ala, Arg, Asn, Asp, Glu, Gly, His, Ile, Leu, Lys,Met, Pro, Ser, Thr, Trp, or Tyr; Xaa₁₅ is Ala, Arg, Asn, Asp, Gly, His,Leu, Phe, Pro, Ser, Trp, or Tyr; Xaa₁₆ is Ala, Asn, Asp, Gly, His, Leu,Phe, Pro, Ser, Trp, or Tyr; and Xaa₁₇ is Ala or Asp.

[0117] In a further embodiment, the invention provides a non-naturallyoccurring, serum albumin-binding agent comprising a linear polypeptidecomprising an amino acid sequence selected from the group consisting of:(SEQ ID NO:104) P T V V Q P K F H A F T H E D L L W I F, (SEQ ID NO:105)L K S Q M V H A L P A A S L H D Q H E L, and (SEQ ID NO:106) S Q V Q G TP D L Q F T V R D F I Y M F.

[0118] Examples of serum albumin-binding agents include polypeptidesthat include an amino acid sequence selected from the group consistingof (depicted using the standard single letter abbreviations for thetwenty common L-amino acids): C T I F L C, (SEQ ID NO:7) C E G K D M I DW V Y C, (SEQ ID NO:8) C D R I A W Y P Q H L C, (SEQ ID NO:9) C D R I AW Y P Q H A C, (SEQ ID NO:41) C D R I A W Y P Q A L C, (SEQ ID NO:42) CD R I A W Y P A H L C, (SEQ ID NO:43) C D R I A W Y A Q H L C, (SEQ IDNO:44) C D R I A W A P Q H L C, (SEQ ID NO:45) C D R I A A Y P Q H L C,(SEQ ID NO:46) C D R A A W Y P Q H L C, (SEQ ID NO:47) C D A I A W Y P QH L C, (SEQ ID NO:48) C A R I A W Y P Q H L C, (SEQ ID NO:49) C E P W ML R F G C, (SEQ ID NO:10) C D Q W F C, (SEQ ID NO:11) C N N A L C, (SEQID NO:12) C D H F F C, (SEQ ID NO:13) C W H F S C, (SEQ ID NO:14) C V TR W A N R D Q Q C, (SEQ ID NO:15) C V T D W A N R H Q H C, (SEQ IDNO:16) C V K D W A N R R R G C, (SEQ ID NO:17) C K F S W I R S P A F C,(SEQ ID NO:18) C Q T T W P F T M M Q C, (SEQ ID NO:107) C V T M W P F EQ I F C, (SEQ ID NO:108) C F T Y Y P F T T F S C, (SEQ ID NO:109) C W TK F P F D L V W C, (SEQ ID NO:110) C V S Y W P H F V P V C, (SEQ IDNO:111) C Y I S F P F D Q M Y C, (SEQ ID NO:112) C S V Q Y P F E V V VC, (SEQ ID NO:113) C W T Q Y P F D H S T C, (SEQ IID NO:114) C I T W P FK R P W P C, (SEQ ID NO:115) C I S W P F E M P F H C, (SEQ ID NO:116) CI T W P F K R P W P C, (SEQ ID NO:117) C I T Y P F H E M F P C, (SEQ IDNO: 118) C I T W P F Q T S Y P C, (SEQ ID NO:119) C K F S W I R S P A FC, (SEQ ID NO:120) C W I V D E D G T K W C, (SEQ ID NO:121) C D S A Y WQ E I P A C, (SEQ ID NO:122) C L W D P M L C, (SEQ ID NO:123) C E H P YW T E V D K C, (SEQ ID NO:124) C D T P Y W R D L W Q C, (SEQ ID NO:125)C Q L P Y M S T P E F C, (SEQ ID NO:126) C G R G F D K E S I Y C, (SEQID NO:127) C V T Y I G T W E T V C, (SEQ ID NO:128) C T D T N W S W M FD C, (SEQ ID NO:129) C T L E I G T W F V F C, (SEQ ID NO:130) C K I A LF Q H F E V C, (SEQ ID NO:131) C I K L Y G L G H M Y C, (SEQ ID NO:132)C E M Q S I I P W W E C, (SEQ ID NO:133) C V E K Y Y W D V L I C, (SEQID NO:134) C P H G R Y S M F P C, (SEQ ID NO:135) C N V R W T D T P Y WC, (SEQ ID NO:136) C T Y D P I A D L L F C, (SEQ ID NO:137) C M D W P NH R D C, (SEQ ID NO:138) C F P I H L T M F C, (SEQ ID NO:139) C Q T S FT N Y W C, (SEQ ID NO:140) C M E F G P D D C, (SEQ ID NO:141) C S W D PI F C, (SEQ ID NO: 142) C A W D P L V C, (SEQ ID NO: 143) C H I Y D W FC, (SEQ ID NO:144) C L W D P M I C, (SEQ ID NO:145) C S P P G K T C,(SEQ ID NO:146) C T F W Q Y W C, (SEQ ID NO:147) C M F E L P F C, (SEQID NO: 148) C F S K P D Q C, (SEQ ID NO:149) C F Y Q W W G C, (SEQ IDNO:150) C T W D P I F C, (SEQ ID NO:151) C W L Y D C, (SEQ ID NO:152) CD K Y G C, and (SEQ ID NO:153) C S K D T C. (SEQ ID NO: 154)

[0119] Additional examples of serum albumin-binding agents arepolypeptides that include an amino acid sequence selected from the groupconsisting of: A D F C E G K D M I D W V Y C R L Y, (SEQ ID NO:27) F W FC D R I A W Y P Q H L C E F L, (SEQ ID NO:28) F W F C D R I A W Y P Q HL C E F A, (SEQ ID NO:50) F W F C D R I A W Y P Q H L C E A L, (SEQ IDNO:51) F W F C D R I A W Y P Q H L C A F L, (SEQ ID NO:52) F W F C D R IA W Y P Q H A C E F L, (SEQ ID NO:53) F W F C D R I A W Y P Q A L C E FL, (SEQ ID NO:54) F W F C D R I A W Y P A H L C E F L, (SEQ ID NO:55) FW F C D R I A W Y A Q H L C E F L, (SEQ ID NO:56) F W F C D R I A W A PQ H L C E F L, (SEQ ID NO:57) F W F C D R I A A Y P Q H L C E F L, (SEQID NO:58) F W F C D R A A W Y P Q H L C E F L, (SEQ ID NO:59) F W F C DA I A W Y P Q H L C E F L, (SEQ ID NO:60) F W F C A R I A W Y P Q H L CE F L, (SEQ ID NO:61) F W A C D R I A W Y P Q H L C E F L, (SEQ IDNO:62) F A F C D R I A W Y P Q H L C E F L, (SEQ ID NO:63) A W F C D R IA W Y P Q H L C E F L, (SEQ ID NO:64) D W D C V T R W A N R D Q Q C W GP, (SEQ ID NO:29) D W D C V T R W A N R D Q Q C W A L, (SEQ ID NO:30) DW D C V T D W A N R H Q H C W A L, (SEQ ID NO:31) D W Q C V K D W A N RR R G C M A D, (SEQ ID NO:32) R N M C K F S W I R S P A F C A R A, (SEQID NO:33) L R D C Q T T W P F M M Q C P N N, (SEQ ID NO:155) N R E C V TM W P F E Q I F C P W P, (SEQ ID NO:156) L R S C F T Y Y P F T T F S C SP A, (SEQ ID NO:157) L S H C W T K F P F D L V W C D S P, (SEQ IDNO:158) L R M C V S Y W P H F V P V C E N P, (SEQ ID NO:159) L R D C Y IS F P F D Q M Y C S H F, (SEQ ID NO:160) F R H C S V Q Y P F E V V V C PA N, (SEQ ID NO:161) L R N C W T Q Y P F D H S T C S P N, (SEQ IDNO:162) D S M C I T W P F K R P W P C A N, (SEQ ID NO:163) A F M C I S WP F E M P F H C S P D, (SEQ ID NO:164) D S M C I T W P F K R P W P C A NP, (SEQ ID NO:165) W D L C I T Y P F H E M F P C E D G, (SEQ ID NO:166)G G E C I T W P F Q T S Y P C T N G, (SEQ ID NO:167) R N M C K F S W I RS P A F C A R A, (SEQ ID NO:168) F S L C W I V D E D G T K W C L P, (SEQID NO:169) R W F C D S A Y W Q E I P A C A R D, (SEQ ID NO:170) R W Y CL W D P M L C M S D, (SEQ ID NO:171) A W Y C E H P Y W T E V D K C H SS, (SEQ ID NO:172) S D F C D T P Y W R D L W Q C N S P, (SEQ ID NO:173)L P W C Q L P Y M S T P E F C I R P, (SEQ ID NO:174) Y H V C G R G F D KE S I Y C K F L, (SEQ ID NO:175) S F C V T Y I G T W E T V C K R S, (SEQID NO:176) N D G C T D T N W S W M F D C P P L, (SEQ ID NO:177) W R D CT L E I G T W F V F C K G S, (SEQ ID NO:178) S P Y C K I A L F Q H F E VC A A D, (SEQ ID NO:179) R H W C I K L Y G L G H M Y C N R S, (SEQ IDNO:180) D H A C E M Q S I I P W W E C Y P H, (SEQ ID NO:181) P R S C V EK Y Y W D V L I C G F F, (SEQ ID NO:182) F H T C P H G R Y S M F P C D YW, (SEQ ID NO:183) H G W C N V R W T D T P Y W C A F S, (SEQ ID NO:184)Y R V C T Y D P I A D L L F C P F N, (SEQ ID NO:185) R S F C M D W P N HR D C D Y S, (SEQ ID NO:186) F W D C F P I H L T M F C D R F, (SEQ IDNO:187) Y L Y C Q T S F T N Y W C A F H, (SEQ ID NO:188) G L Y C M E F GP D D C A W H, (SEQ ID NO:189) K N F C S W D P I F C G I H, (SEQ IDNO:190) K W Y C A W D P L V C E I F, (SEQ ID NO:191) W T T C H I Y D W FC S S S, (SEQ ID NO:192) Q W Y C L W D P M I C G L I, (SEQ ID NO:193) QT N C S P P G K T C D K N, (SEQ ID NO:194) A I C T F W Q Y W C L E P,(SEQ ID NO:195) F E W C M F E L P F C S W P, (SEQ ID NO:196) Q E G C F SK P D Q C K V M, (SEQ ID NO:197) L E Y C F Y Q W W G C P H A, (SEQ IDNO:198) Y Q F C T W D P I F C G W H, (SEQ ID NO:199) L W D C W L Y D C EG N, (SEQ ID NO:200) V H S C D K Y G C V N A, (SEQ ID NO:201) F E H C SK D T C S G N, (SEQ ID NO:202) V A W C T I F L C L D V, (SEQ ID NO:203)F K I C D Q W F C L M P, (SEQ ID NO:204) H V G C N N A L C M Q Y, (SEQID NO:205) W K V C D H F F C L S P, (SEQ ID NO:206) N H G C W H F S C IW D, (SEQ ID NO:207) F R N C E P W M L R F G C N P R, (SEQ ID NO:208) AD F C E G K D M I D W V Y C R L Y, (SEQ ID NO:209) F W F C D R I A W Y PQ H L C E F L D, (SEQ ID NO:210) D W D C V T R W A N R D Q Q C W G P,(SEQ ID NO:211) D W D C V T R W A N R D Q Q C W A L, (SEQ ID NO:212) D WD C V T D W A N R H Q H C W A L, (SEQ ID NO:213) D W Q C V K D W A N R RR G C M A D, (SEQ ID NO:214) R N M C K F S W I R S P A F C A R A D P,(SEQ ID NO:215).

[0120] Additional examples of serum albumin-binding agents includepolypeptides that comprising an amino acid sequence selected from thegroup consisting of: A E G T G D A D F C E G K D M I D W V Y C R L Y D PE, (SEQ ID NO:34) A E G T G D F W F C D R I A W Y P Q H L C E F L D P E,(SEQ ID NO:35) A E G T G D F W F C D R I A W Y P Q H L C E F L A P E,(SEQ ID NO:65) A E G T G D F W F C D R I A W Y P Q H L C E F A D P E,(SEQ ID NO:66) A E G T G D F W F C D R I A W Y P Q H L C E A L D P E,(SEQ ID NO:67) A E G T G D F W F C D R I A W Y P Q H L C A F L D P E,(SEQ ID NO:68) A E G T G D F W F C D R I A W Y P Q H A C E F L D P E,(SEQ ID NO:69) A E G T G D F W F C D R I A W Y P Q A L C E F L D P E,(SEQ ID NO:70) A E G T G D F W F C D R I A W Y P A H L C E F L D P E,(SEQ ID NO:71) A E G T G D F W F C D R I A W Y A Q H L C E F L D P E,(SEQ ID NO:72) A E G T G D F W F C D R I A W A P Q H L C E F L D P E,(SEQ ID NO:73) A E G T G D F W F C D R I A A Y P Q H L C E F L D P E,(SEQ ID NO:74) A E G T G D F W F C D R A A W Y P Q H L C E F L D P E,(SEQ ID NO:75) A E G T G D F W F C D A I A W Y P Q H L C E F L D P E,(SEQ ID NO:76) A E G T G D F W F C A R I A W Y P Q H L C E F L D P E,(SEQ ID NO:77) A E G T G D F W A C D R I A W Y P Q H L C E F L D P E,(SEQ ID NO:78) A E G T G D F A F C D R I A W Y P Q H L C E F L D P E,(SEQ ID NO:79) A E G T G D A W F C D R I A W Y P Q H L C E F L D P E,(SEQ ID NO:80) A E G T G A F W F C D R I A W Y P Q H L C E F L D P E,(SEQ ID NO:81) A E G T G D D W D C V T R W A N R D Q Q C W G P D P E,(SEQ ID NO:36) A E G T G D D W D C V T R W A N R D Q Q C W A L D P E,(SEQ ID NO:37) A E G T G D D W D C V T D W A N R H Q H C W A L D P E,(SEQ ID NO:38) A E G T G D D W Q C V K D W A N R R R G C M A D D P E,and (SEQ ID NO:39) A E G T G D R N M C K F S W I R S P A F C A R A D PE. (SEQ ID NO:40)

[0121] Particular examples of a serum albumin-binding agents arepolypeptides that include a compound of the formula:

[0122] AEGTGDFWFCDRIAWYPQHLCEFLDPEGGGK(SEQ ID NO: 19). This polypeptideis designated DX-236.

[0123] DX-236 binds mammalian serum albumins and is useful underappropriate conditions as a “pan mammalian” serum albumin-binding agent.DX-236 variants that include between one and five amino acid changes(substitutions, insertions, or deletions), e.g., between one and three,or one or two,; or between one and six conservative amino acidsubstitutions, e.g., between one and four, one and three, or one andtwo; and that bind to a serum albumin can also be used. The followingtwo DX-236 variants can be used: DX-236A which includes the peptidesequence: FWFCDRIAWYPQHLCEFLD (SEQ ID NO: 210) and DX-236B whichincludes the peptide sequence:

[0124] CDRIAWYPQHLC (SEQ ID NO: 9)

[0125] DX-236 can also include additional chemical modifications, forexample:

[0126] Ac-AEGTGDFWFCDRIAWYPQHLCEFLDPEGGGK—NH₂ (SEQ ID NO: 19), whereinAc indicates an N-terminal acetyl capping group and —NH₂ indicates aC-terminal amide capping group. Examples of DX-236 variants includecompounds that include the following sequences:

[0127] AEGTGDFWFCDRIAWYPQHLCEFLAPEGGGK—,

[0128] AEGTGDFWFCDRIAWYPQHLCEFADPEGGGK—,

[0129] AEGTGDFWFCDRIAWYPQHLCEALDPEGGGK—,

[0130] AEGTGDFWFCDRIAWYPQHLCAFLDPEGGGK—,

[0131] AEGTGDFWFCDRIAWYPQHACEFLDPEGGGK—,

[0132] AEGTGDFWFCDRIAWYPQALCEFLDPEGGGK—,

[0133] AEGTGDFWFCDRIAWYPAHLCEFLDPEGGGK—,

[0134] AEGTGDFWFCDRIAWYAQHLCEFLDPEGGGK—,

[0135] AEGTGDFWFCDRIAWAPQHLCEFLDPEGGGK—,

[0136] AEGTGDFWFCDRIAAYPQHLCEFLDPEGGGK—,

[0137] AEGTGDFWFCDRAAWYPQHLCEFLDPEGGGK—,

[0138] AEGTGDFWFCDAIAWYPQHLCEFLDPEGGGK—,

[0139] AEGTGDFWFCARIAWYPQHLCEFLDPEGGGK—,

[0140] AEGTGDFWACDRIAWYPQHLCEFLDPEGGGK—,

[0141] AEGTGDFAFCDRIAWYPQHLCEFLDPEGGGK—,

[0142] AEGTGDAWFCDRIAWYPQHLCEFLDPEGGGK—, and

[0143] AEGTGAFWFCDRIAWYPQHLCEFLDPEGGGK—,

[0144] (SEQ ID NOs: 82 through 98, respectively). The variants canfurther include an N— or C-terminal modification. Exemplary variantshave between one and six, one and five, one and four, or one and threeamino acid substitutions, e.g., one or two amino acid substitutions.Other variants include one, two, three, or less than five amino acidinsertions, deletions, or substitutions.

[0145] Additional serum albumin-binding agents include the following:

[0146] GDLRDCQTTWPFTMMQCPNNDPGGGK—,

[0147] GDNRECVTMWPFEQIFCPWPDPGGGK—,

[0148] GDLRSCFTYYPFTTFSCSPADP GGGK—,

[0149] GDDSMCITWPFKRPWPCANDPGGGK—,

[0150] GDRNMCKFSWIRSPAFCARADPGGGK—,

[0151] GDFSLCWIVDEDGTKWCLPDPGGGK—,

[0152] GDRWFCDSAYWQEIPACARDDPGGGK—,

[0153] GDSDFCDTPYWRDLWQCNSPDPGGGK—,

[0154] GDSFCVTYIGTWETVCKRSDPGGGK—,

[0155] GDNDGCTDTNWSWMFDCPPLDPGGGK—,

[0156] GDSPYCKIALFQHFEVCAADDPGGGK—,

[0157] GDPRSCVEKYYWDVLICGFFDPGGGK—,

[0158] GSRSFCMDWPNHRDCDYSAPGGGK—,

[0159] AGKWYCAWDPLVCEIFGTGGGK—,

[0160] AGWTTCHIYDWFCSSSGTGGGK—,

[0161] AGLEYCFYQWWGCPHAGTGGGK—,

[0162] AGYQFCTWDPIFCGWHGTGGGK—, and

[0163] GSLWDCWLYDCEGNAPGGGK—,

[0164] (SEQ ID NOs: 216 through 233, respectively).

[0165] Another particular serum albumin-binding agent is a compound thatincludes: AEGTGDRNMCKFSWIRSPAFCARADPE (SEQ ID NO: 20). This bindingmoiety is designated polypeptide compound DX-321. Dx-321 can also bemodified, e.g., as follows:

[0166] Ac-AEGTGDRNMCKFSWIRSPAFCARADPE-X—K—NH₂ (SEQ ID NO: 24), whereinAc indicates an N-terminal acetyl capping group, X (above) indicates apolypeptide linked 6-aminohexanoic acid group, and —NH₂ indicates aC-terminal amide capping group. DX-321 preferentially binds human serumalbumin (HSA) over serum albumins from other species under appropriateconditions. DX-321 is useful as a reagent to specifically detect orisolate HSA. In some embodiments, the compounds do not include theN-terminal acetyl capping group, and may or may not include a C-terminalamide capping group.

[0167] DX-321 variants that include between one and five amino acidchanges (substitutions, insertions, or deletions), e.g., between one andthree, or one or two,; or between one and six conservative amino acidsubstitutions, e.g., between one and four, one and three, or one andtwo; and that bind to a serum albumin can also be used. The followingDX-321 variants can also be used: DX-321-A which includes the peptidesequence: RNMCKFSWIRSPAFCARA (SEQ ID NO: 430); and DX-321-B whichincludes the peptide sequence: CKFSWIRSPAFC (SEQ ID NO: 120).

[0168] Examples of specific immunoglobulin binding molecules (which bindthe Fc region of immunoglobulin) include polypeptides comprising aminoacid sequences of the following four general formulae:

Z1-X1-X2-X3-X4-W—C-Z2 (SEQ ID NO: 234);   I.

[0169] wherein, Z1 is a polypeptide of at least 6 amino acids; X1 is G,H, N, R, or S;

[0170] X2 is A, D, E, F, I, M, or S; X3 is A, I, L, M, or V; X4 is I, M,T, or V; Z2 is a polypeptide of at least one amino acid or is absent;and Z1 contains at least one cysteine residue such that formation of adisulfide bond with the invariant cysteine residue forms a cyclicpeptide of 12 amino acids.

Z1-X—W-Z2-W-Z3 (SEQ ID NO: 235)   II.

[0171] wherein, Z1 is a polypeptide of at least one amino acid or isabsent;

[0172] X is F or Y; Z2 is a tripeptide; and Z3 is a polypeptide of atleast one amino acid; and

[0173] wherein at least two of the polypeptides Z1, Z2, and Z3 contain acysteine residue, such that formation of a disulfide bond between suchcysteine residues forms a cyclic peptide of 7-12 amino acids.

[0174] In the foregoing formula II polypeptides, Z2 can have the formula(IIA):

X1-X2-X3   (IIA),

[0175] wherein, X1 is A, C, F, K, P, R, W, or Y; X2 is C, D, E, G, H, K,M, N, Q, R, S, T, V, or Y; and X3 is A, E, F, H, I, K, L, Q, R, S, T, V,or Y; with the proviso that at most one of X1, X2 and X3 can be C. Insome implementations, where X2 is C, then X1 is Y. In someimplementations, X1 is C.

Z1-W-Z2-W—W-Z3 (SEQ ID NO: 236);   III.

[0176] wherein, Z1 is a polypeptide of at least one amino acid; Z2 is atripeptide; and Z3 is a polypeptide of at least one amino acid; whereinat least two of the polypeptides Z 1, Z2, and Z3 contain a cysteineresidue, such that formation of a disulfide bond between such cysteineresidues forms a cyclic peptide of 8-12 amino acids, with the provisothat where Z1 contains a cysteine, then Z2 does not contain a cysteine,and where Z2 contains a cysteine, it is the middle residue of thetripeptide and Z3 also contains a cysteine.

[0177] In some cases, for the polypeptides of formula III, when Z1 andZ3 each contain a cysteine residue, the cysteine of Z1 is adjacent theinvariant tryptophan (W), the first amino acid of Z2 is lysine and thesecond amino acid of Z3 is aspartic acid (D).

Z1-P—X1-W—X2-C—X3-X4-X5 (SEQ ID NO: 237);   IV.

[0178] wherein, Z1 is a polypeptide of at least one amino acid andincludes a cysteine residue; X1 is A, E, R, S, or T; X2 is F, W, or Y;X3 is D, E, L, M, or Q; X4 is H, W, or Y;

[0179] X5 is F or Y; and wherein the cysteine residue in Z1 and thecysteine residue between X2 and X3 form a cyclic peptide of 10-12 aminoacids.

[0180] Examples of immunoglobulin binding polypeptides includepolypeptides comprising amino acid sequences selected from the groupconsisting of: R-R-A-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H (SEQ ID NO:238)W-G-E-C-T-V-T-S-Y-G-E-L-I-W-C-G-G-L (SEQ ID NO:239)S-S-A-C-A-F-D-P-M-G-A-V-W-C-T-Y-D (SEQ ID NO:240)L-L-E-C-A-Y-N-T-S-G-E-L-I-W-C-N-G-S (SEQ ID NO:241)P-D-D-C-S-I-H-F-S-G-E-L-I-W-C-E-P-L (SEQ ID NO:242)L-G-E-C-T-V-T-S-Y-G-E-L-I-W-C-G-G-L (SEQ ID NO:243)W-G-E-C-T-V-T-S-Y-G-E-L-I-W-C-G-G-H (SEQ ID NO:244)D-H-M-C-V-Y-T-T-W-G-E-L-I-W-C-D-D-H (SEQ ID NO:245)W-G-E-C-T-V-T-S-Y-G-E-L-I-W-C-G-G-L (SEQ ID NO:246)C-R-A-C-S-R-D-W-P-G-A-L-V-W-C-A-G-H (SEQ ID NO:247)R-R-A-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H (SEQ ID NO:248)L-H-A-C-A-F-D-P-M-G-A-V-I-W-C-T-Y-D (SEQ ID NO:249)D-H-M-C-V-Y-T-T-W-G-E-L-M-W-C-D-N-H (SEQ ID NO:250)P-P-T-C-T-W-D-W-Q-G-I-L-V-W-C-S-G-H (SEQ ID NO:251)S-N-K-C-S-N-T-W-D-G-S-L-I-W-C-S-A-N (SEQ ID NO:252)F-P-E-C-T-F-D-M-E-G-F-L-I-W-C-S-S-F (SEQ ID NO:253)H-D-L-C-A-Q-A-P-F-G-D-A-T-W-C-D-L-R (SEQ ID NO:254)P-N-H-C-S-Y-N-L-K-S-E-L-I-W-C-Q-D-L (SEQ ID NO:255)P-L-D-C-A-R-D-I-H-N-S-L-I-W-C-S-L-G (SEQ ID NO:256)G-S-E-C-S-W-T-S-L-N-E-L-I-W-C-A-H-W (SEQ ID NO:257)W-P-D-C-S-F-T-V-Q-R-D-L-I-W-C-E-A-L (SEQ ID NO:258)S-H-S-C-A-Y-D-Y-A-H-M-L-V-W-C-T-H-F (SEQ ID NO:259)D-H-M-C-V-Y-T-T-W-G-E-L-I-W-C-D-N-H (SEQ ID NO:260)R-P-N-C-T-F-A-A-S-G-E-L-I-W-C-M-H-Y (SEQ ID NO:261)W-W-G-C-Q-F-D-W-R-G-E-L-V-W-C-P-Y-L (SEQ ID NO:262)G-G-V-C-S-Y-S-G-M-G-E-I-V-W-C-R-W-F (SEQ ID NO:263)A-L-M-C-S-H-D-M-W-G-S-L-I-W-C-K-H-F (SEQ ID NO:264)W-W-N-C-H-N-G-W-T-W-T-G-G-W-C-W-W-F (SEQ ID NO:265)Y-H-V-C-A-R-D-S-W-D-Q-L-I-W-C-E-A-F (SEQ ID NO:266)N-Y-W-C-N-F-W-Q-L-P-T-C-D-N-L (SEQ ID NO:267)Y-W-Y-C-K-W-F-S-E-S-A-S-C-S-S-R (SEQ ID NO:268)Y-W-Y-C-K-W-F-E-D-K-H-P-C-D-S-S (SEQ ID NO:269)Y-W-Y-C-S-W-F-P-D-R-P-D-C-P-L-Y (SEQ ID NO:270)N-Y-W-C-N-V-W-L-L-G-D-V-C-R-S-H (SEQ ID NO:271)L-Y-W-C-H-V-W-F-G-Q-H-A-W-Q-C-K-Y-P (SEQ ID NO:272)Y-W-K-C-K-W-M-P-W-M-C-G-F-D (SEQ ID NO:273)D-D-H-C-Y-W-F-R-E-W-F-N-S-E-C-P-H-G (SEQ ID NO:274)N-Y-W-C-N-I-W-G-L-H--G-C-N-S-H (SEQ ID NO:275)Y-W-F-C-Q-W-F-S-Q-N-H-T-C-F-R-D (SEQ ID NO:276)H-Y-W-C-D-I-W-F-G-A-P-A-C-Q-F-R (SEQ ID NO:277)S-G-D-C-G-F-W-P-R-I-W-G-L-C-M-D-N (SEQ ID NO:278)F-W-Y-C-K-W-F-Y-E-D-A-Q-C-S-H-D (SEQ ID NO:279)Y-Y-W-C-N-Y-W-G-L-C-P-D-Q (SEQ ID NO:280) S-Y-W-C-K-I-W-D-V-C-P-Q-S (SEQID NO:281) K-Y-W-C-N-L-W-G-V-C-P-A-N (SEQ ID NO:282)Q-Y-W-C-Y-Q-W-G-L-C-G-A-N (SEQ ID NO:283) K-Y-W-C-Q-Q-W-G-V-C-N-G-S (SEQID NO:284) K-Y-W-C-V-Q-W-G-V-C-P-E-S (SEQ ID NO:285)K-Y-W-C-M-Q-W-G-L-C-G-W-E (SEQ ID NO:286) H-F-W-C-E-V-W-G-L-C-P-S-I (SEQID NO:287) Q-Y-W-C-T-K-W-G-L-C-T-N-V (SEQ ID NO:288)A-Y-W-C-K-V-W-G-L-C-Q-G-E (SEQ ID NO:289) K-Y-W-C-N-L-W-G-V-C-P-A-N (SEQID NO:290) Q-Y-W-C-N-V-W-G-V-C-L-P-S (SEQ ID NO:291)H-Y-W-C-Q-Q-W-G-I-C-E-R-P (SEQ ID NO:292) R-Y-W-C-N-I-W-D-V-C-P-E-Q (SEQID NO:293) Q-Y-W-C-T-H-W-G-L-C-G-K-Y (SEQ ID NO:294)T-Y-W-C-T-K-W-G-L-C-P-H-N (SEQ ID NO:295) F-Y-W-C-G-Q-W-G-L-C-A-P-P (SEQID NO:296) G-Y-W-C-N-V-W-G-L-C-S-T-E (SEQ ID NO:297)R-Y-W-C-G-V-W-G-V-C-E-I-D (SEQ ID NO:298) K-F-W-C-T-I-W-G-V-C-H-M-P (SEQID NO:299) H-Y-W-C-Q-Q-W-G-I-C-E-R-P (SEQ ID NO:300)R-Y-W-C-N-I-W-D-V-C-P-E-Q (SEQ ID NO:301) F-Y-W-C-S-Q-W-G-L-C-K-Y-D (SEQID NO:302) H-Y-W-C-E-K-W-G-L-C-L-M-S (SEQ ID NO:303)H-Y-W-C-Q-K-W-G-V-C-P-T-D (SEQ ID NO:304) H-Y-W-C-S-L-W-G-V-C-D-I-N (SEQID NO:305) R-F-W-C-S-A-W-G-V-C-P-A (SEQ ID NO:306)S-Y-W-C-K-I-W-D-V-C-P-Q-S (SEQ ID NO:307) Q-Y-W-C-S-I-W-K-V-C-P-G-R (SEQID NO:308) Y-W-Y-C-E-W-F-G-A-C-I-N-D (SEQ ID NO:309)E-Y-W-C-K-Y-W-G-L-E-C-V-H-R (SEQ ID NO:310) K-Y-W-C-T-Q-W-G-L-K-C-D-K-Q(SEQ ID NO:311) K-Y-W-C-S-F-W-G-L-Q-C-K-T (SEQ ID NO:312)R-Y-W-C-N-F-W-G-V-N-C-D-A-N (SEQ ID NO:313) N-Y-W-C-T-H-W-G-V-M-C-L-D-H(SEQ ID NO:314) Y-W-F-C-K-W-F-P-S-Q-C-Q-F-M (SEQ ID NO:315)A-Y-W-C-K-Q-W-G-L-K-C-Q-L-G (SEQ ID NO:316) K-Y-W-C-K-F-W-G-L-E-C-K-V-G(SEQ ID NO:317) N-Y-W-C-T-E-W-G-L-N-C-N-N-K (SEQ ID NO:318)S-Y-W-C-E-K-W-G-L-T-C-E-T-H (SEQ ID NO:319) E-Y-W-C-R-I-W-G-L-Q-C-N-M-V(SEQ ID NO:320) K-Y-W-C-K-K-W-G-V-N-C-D-F-N (SEQ ID NO:321)K-Y-W-C-S-V-W-G-V-Q-C-P-H-S (SEQ ID NO:322) F-Y-W-C-T-K-W-G-L-E-C-I-H-S(SEQ ID NO:323) H-Y-W-C-Q-Q-W-G-L-M-C-F-E-T (SEQ ID NO:324)K-Y-W-C-K-R-W-G-L-M-C-N-G-G (SEQ ID NO:325) A-Y-W-C-M-T-W-G-V-P-C-I-S-W(SEQ ID NO:326) K-Y-W-C-K-K-W-G-V-N-C-D-F-N (SEQ ID NO:327)K-Y-W-C-S-V-W-G-V-Q-C-P-D-S (SEQ ID NO:328) K-Y-W-C-S-V-W-G-V-Q-C-P-H-S(SEQ ID NO:329) L-Y-W-C-T-K-W-G-V-T-C-Q-K-D (SEQ ID NO:330)T-Y-W-C-H-K-W-G-V-K-C-A-T-T (SEQ ID NO:331) T-Y-W-C-T-F-W-E-L-P-C-D-P-A(SEQ ID NO:332) K-Y-W-C-T-K-W-Q-L-N-C-E-E-V (SEQ ID NO:333)N-Y-W-C-H-F-W-Q-V-P-C-L-E-Q (SEQ ID NO:334) T-Y-W-C-V-V-W-N-V-P-C-S-T-D(SEQ ID NO:335) N-F-W-C-H-T-W-G-L-Q-C-N-D-L (SEQ ID NO:336)F-W-Y-C-Y-W-F-N-E-K-C-K-T-P (SEQ ID NO:337) G-F-W-C-T-F-W-G-V-T-C-E-A-G(SEQ ID NO:338) P-H-N-C-D-D-H-Y-W-Y-C-K-W-F (SEQ ID NO:339)E-M-T-C-S-S-H-Y-W-Y-C-T-W-M (SEQ ID NO:340) H-I-D-C-K-T-N-Y-W-W-C-R-W-T(SEQ ID NO:341) E-M-R-C-G-Q-H-F-W-Y-C-E-W-F (SEQ ID NO:342)N-Y-W-C-N-F-W-Q-L-P-T-C-D-N-L (SEQ ID NO:343)Y-W-Y-C-Q-W-F-Q-E-V-N-K-C-F-N-S (SEQ ID NO:344)Y-Y-W-C-R-H-W-F-P-D-F-D-C-V-H-S (SEQ ID NO:345)Y-W-Y-C-S-W-F-P-D-R-P-D-C-P-L-Y (SEQ ID NO:346)Y-W-Y-C-V-W-F-D-N-A-D-Q-C-V-H-H (SEQ ID NO:347)A-A-T-C-S-T-S-Y-W-Y-Y-Q-W-F-C-T-D-S (SEQ ID NO:348)Y-W-A-C-V-W-G-L-K-S-C-V-D-R (SEQ ID NO:349) Y-W-R-C-V-W-F-P-A-S-C-P-T(SEQ ID NO:350) D-W-Q-C-L-W-W-G-N-S-F-W-P-Y-C-A-N-L (SEQ ID NO:351)F-W-R-C-H-W-W-P-E-R-C-P-V-D (SEQ ID NO:352)N-P-M-C-W-K-K-S-W-W-E-D-A-Y-C-I-N-H (SEQ ID NO:353)S-W-V-C-W-K-A-K-W-W-E-D-K-R-C-A-P-F (SEQ ID NO:354)S-R-Q-C-W-K-E-L-W-W-T-D-Q-M-C-L-D-L (SEQ ID NO:355)S-F-R-C-Q-S-S-F-P-S-W-Y-C-D-Y-Y (SEQ ID NO: 356)S-W-H-C-Q-N-T-Y-P-E-W-Y-C-Q-W-Y (SEQ ID NO:357)G-S-K-C-K-Q-T-G-F-P-R-W-W-C-E-H-Y (SEQ ID NO:358)D-G-V-C-G-P-R-G-F-G-P-A-W-F-C-M-H-Y (SEQ ID NO:359)Y-S-H-C-A-T-H-Y-P-T-W-Y-C-L-H-F (SEQ ID NO:360)F-C-N-C-W-G-S-H-E-F-T-F-C-V-D-D (SEQ ID NO:361)P-G-W-C-Y-S-D-I-W-G-F-K-H-F-C-N-L-D (SEQ ID NO:362)D-S-S-C-I-K-H-H-N-K-V-T-C-F-F-P (SEQ ID NO:363)R-W-S-C-W-G-V-W-G-C-V-W-V (SEQ ID NO:364) P-V-D-C-K-H-H-F-W-W-C-Y-W-N(SEQ ID NO:365) S-W-N-C-A-F-H-H-N-E-M-V-W-C-D-D-G (SEQ ID NO:366)Y-W-Y-C-W-F-P-D-R-P-E-C-P-L-Y (SEQ ID NO:367)N-P-M-C-W-R-A-S-W-W-E-D-A-Y-C-I-N-H (SEQ ID NO:409)N-P-M-C-W-R-A-H-W-W-E-D-A-Y-C-I-N-H (SEQ ID NO:410)E-H-M-C-V-Y-T-T-W-G-E-L-I-W-C-D-N-H (SEQ ID NO:411)A-C-V-Y-T-T-W-G-E-L-I-W-C-D-N-H (SEQ ID NO:412)T-C-V-Y-T-T-W-G-E-L-I-W-C-D-N-H (SEQ ID NO:413)E-C-V-Y-T-T-W-G-E-L-I-W-C-D-N-H (SEQ ID NO:414)V-C-V-Y-T-T-W-G-E-L-I-W-C-D-N-H (SEQ ID NO:415)[Nle]-C-V-Y-T-T-W-G-E-L-I-W-C-D-N-H (SEQ ID NO:416)S-R-A-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H (SEQ ID NO:417)E-R-A-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H (SEQ ID NO:418)A-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H (SEQ ID NO:419)T-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H (SEQ ID NO:420)E-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H (SEQ ID NO:421)V-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H and (SEQ ID NO:422)G-C-S-R-D-W-S-G-A-L-V-W-C-A-G-H. (SEQ ID NO:423)

[0181] N-terminal and/or C-terminal truncations of the above Fc-regionbinding polypeptides can also be used, particularly cyclic polypeptidesthat retain binding affinity for antibody Fc-regions.

[0182] Fc-region binding molecules according to the above formulae caninclude the following: polypeptides of formula I, in which X1 is G; X2is A or E; X3 is L; and X4 is I or V; polypeptides of formula II, inwhich X is F or Y; and in the tripeptide of formula IIA, X1 is C or Y;X2 is C, K, N or T; and X3 is F, I, K, Q or V.

[0183] Particular examples of immunoglobulin binding molecules includeproteins that include the following polypeptides: RRACSRDWSGALVWCAGH;(SEQ ID NO:238) DHMCVYTTWGELIWCDNH; (SEQ ID NO:260) KYWCSFWGLQCKT; (SEQID NO:312) PVDCKHHFWWCYWN; (SEQ ID NO:365) DDHCYWFREWFNSECPHG; (SEQ IDNO:274) YYWCNYWGLCPDQ; (SEQ ID NO:280) PHNCDDHYWYCKWF; (SEQ ID NO:339)SYWCKIWDVCPQS; (SEQ ID NO:281) KYWCNLWGVCPAN; (SEQ ID NO:282)AATCSTSYWYYQWFCTDS; (SEQ ID NO:348) TYWCTFWELPCDPA; (SEQ ID NO:332)YWYCWFPDRPECPLY; (SEQ ID NO:367) SWVCWKAKWWEDKRCAPF; (SEQ ID NO:354)NPMCWKKSWWEDAYCINH; and (SEQ ID NO:353) SWNCAFHHNEMVWCDDG. (SEQ IDNO:366)

[0184] Still other exemplary polypeptides can have the followingsequences, and may include optional amino-terminal (e.g., acetylation)and carboxy-tenninal modifications (e.g., amidation):GDDHMCVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:368, designated DX249)AGKYWCSFWGLQCKTGTPGPEGGGK; (SEQ ID NO:370, designated DX250)AGPVDCKHHFWWCYWNGTPGPEGGGK; (SEQ ID NO:377, designated DX251)GDDDHCYWFREWFNSECPHGEPGPEGGGK; (SEQ ID NO:378, designated DX252)GDRRACSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:369, designated DX253)AGYYWCNYWGLCPDQGTPGPEGGGK; (SEQ ID NO:379, designated DX254)AGPHNCDDHYWYCKWFPGPEGGGK; (SEQ ID NO374, designated DX389)AGSYWCKIWDVCPQSPGPEGGGK; (SEQ ID NO:371, designated DX392)AGKYWCNLWGVCPANPGPEGGGK; (SEQ ID NO:372, designated DX395)AGAATCSTSYWYYQWFCTDSPGPEGGGK; (SEQ ID NO:375, designated DX398)AGTYWCTFWELPCDPAPGPEGGGK; (SEQ ID NO:373, designated DX404)AGYWYCWFPDRPECPLYPGPEGGGK; (SEQ ID NO:376, designated DX413)GDSWVCWKAKWWEDKRCAPFGTPGPEGGGK; (SEQ ID NO:380, designated DX595)GDNPMCWKKSWWEDAYCINHGTPGPEGGGK; (SEQ ID NO:381, designated DX596)GDSWNCAFHHNEMVWCDDGGTPGPEGGGK; (SEQ ID NO:382, designated DX597)GDWGECTVTSYGELIWCGGLEPGPEGGGK; (SEQ ID NO:383, designated DX1070)GDNPMCWRASWWEDAYCINHEPGPEGGGK; (SEQ ID NO:384, designated DX1071)GDNPMCWRAHWWEDAYCINHEPGpPGGGK; (SEQ ID NO:385, designated DX1072)GDDHMCVYTTWGELIWCDNHEPGPEG-J-NH2 (SEQ ID NO:386, designated DX877)GDDHMCVYTTWGELIWCDNHEPG-J-Su-J-NH2 (SEQ ID NO:387, designated DX878)GDDHMCVYTTWGELIWCDNHEPG-J-Z-J-NH2 (SEQ ID NO:388, designated DX905)GDDHMCVYTTWGELIWCDNH-J-NH2 (SEQ ID NO:389, designated DX907)GDDHMCVYTTWGELIWCDNH-J-Su-J-NH2 (SEQ ID NO:390, designated DX909)GDDHMCVYTTWGELIWCDNH-J-Z-J-NH2 (SEQ ID NO:391, designated DX911)DHMCVYTTWGELIWCDNHEPEGGGK; (SEQ ID NO:392, designated DX1062)EHMCVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:393, designated DX1063)ACVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:394, designated DX1064)TCVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:395, designated DX1065)ECVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:396, designated DX1066)VCVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:397, designated DX1067)Ac-[Nle]CVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:398, designated DX1068)CVYTTWGELIWCDNHEPGPEGGGK; (SEQ ID NO:399, designated DX1069)SRACSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:400, designated DX1139)RRACSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:401, designated DX1142)ERACSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:402, designated DX1141)ACSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:403, designated DX1142)TCSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:404, designated DX1143)ECSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:405, designated DX1144)VCSRDWSGALVWCAGHEPGPEGGGK; (SEQ ID NO:406, designated DX1145)GCSRDWSGALVWCAGHEPGPEGGGK; and (SEQ ID NO:407, designated DX1146)CSRDWSGALVWCAGHEPGPEGGGK-NH2. (SEQ ID NO:408, designated DX1147)

[0185] With respect to the foregoing polypeptides, the polypeptides canfurther include a chemical modification, e.g., N-terminal acetylationand/or C-terminal amidation: e.g., one of the following: -J-NH₂ denotesthe C-terminal group —NH—(CH₂CH₂O)₂—CH₂CH₂—NH₂, -J-Su-J-NH₂ denotes theC-terminal group—NH—(CH₂CH₂O)₂—CH₂CH₂—NH—C:O—CH₂CH₂—C:O—NH(CH₂CH₂O)₂—CH₂CH₂—NH₂,-J-Z-J-NH₂ denotes the C-terminal group—NH—(CH₂CH₂O)₂—CH₂CH₂—NH—C:O—CH₂—O—(CH₂CH₂O)₂—CH₂—C:ONH—(CH₂CH₂O)₂—CH₂CH₂—NH₂, and [Nle] denotes norleucine.

[0186] The immunoglobulin binding polypeptides can have high affinity(e.g., K_(D) in the range 10 μM to 0.01 μM, more preferably in the range1.0 μM to 0.01 μM) for human Fc polypeptides or particular IgG isotypes(e.g., IgGI, IgG2, IgG3 and/or IgG4). Some polypeptides also showspecies specificity (e.g., binding to human but not other mammalianIgGs). For example:

[0187] DX249 exhibits dissociation constants (K_(D)) for human IgGI ofless than 0.1 μM at pH 5.7 and less than 0.5 μM at pH 7.4;

[0188] DX252 exhibits dissociation constants (K_(D)) for human IgG3 ofless than 0.1 μM at pH 5.7 and in the range of 2.1 μM to 3.4 μM forIgGI, IgG2, IgG3, and IgG4 at pH 7.4;

[0189] DX253 exhibits quantitative binding of Fc protein (captureefficiency >90% of total load) from buffer solution and tobacco extract;

[0190] DX254 exhibits dissociation constants (K_(D)) for human IgG1 ofless than 0.1 μM at pH 5.7, less than 2.0 μM at pH 7.4, and less than1.0 μM at pH 9.3;

[0191] DX301 exhibits dissociation constants below about 10 μM for humanFe, IgG1, IgG2 and IgG4; and

[0192] DX300 exhibits a dissociation constant of 4.1±4.6 for human IgG3.Variants of the above peptides can also be used, including the segmentDX249-A, DHMCVYTTWGELIWCDNH (SEQ ID NO: 260); the segment DX253-A,RRACSRDWSGALVWCAGH (SEQ ID NO: 238); and AATCSTSYWYYQWFCTDS (SEQ ID NO:348).

[0193] The term “associated” refers to a direct or indirect physicalattachment between compounds. Attachments can be mediated by a covalentor non-covalent interaction. An indirectly physical attachment refersto, for example, a case where two compounds are not in direct contactwith each other, but each contact one or more intermediary compounds.

[0194] The term “polypeptide” refers to a polymer of three or more aminoacids linked by a peptide bond. The polypeptide may include one or moreunnatural amino acids. Typically, the polypeptide includes only naturalamino acids. The term “peptide” refers to a polypeptide that is betweenthree and thirty-two amino acids in length. A protein can include one ormore polypeptide chains.

[0195] The term “antibody” as used herein refers to an immunoglobulinmolecule or immunologically active portion thereof, i.e., anantigen-binding portion. An antibody can include at least one, andpreferably two, heavy (H) chain variable regions (abbreviated herein asVH), and at least one and preferably two light (L) chain variableregions (abbreviated herein as VL). The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). The extent of the frameworkregion and CDR's has been precisely defined (see, Kabat, E. A., et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition,U.S. Department of Health and Human Services, NIH Publication No.91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). EachVH and VL is composed of three CDR's and four FRs, arranged fromamino-terminus to carboxy-terminus in the following order: FR1, CDR1,FR2, CDR2, FR3, CDR3, FR4.

[0196] As used herein, the term “immunoglobulin” refers to a proteinconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes. Some human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 KDa or 214 amino acids) are encoded by a variableregion gene at the NH₂-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH—terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 KDa or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

[0197] The term “antigen-binding fragment” of an antibody (or simply“antibody portion,” or “fragment”), as used herein, refers to one ormore fragments of a full-length antibody that retain the ability tospecifically bind to the antigen. Examples of antigen-binding fragmentsinclude, but are not limited to: (i) a Fab fragment, a monovalentfragment consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab′)₂fragment, a bivalent fragment comprising two Fab fragments linked by adisulfide bridge at the hinge region; (iii) a Fd fragment consisting ofthe VH and CH1 domains; (iv) a Fv fragment consisting of the VL and VHdomains of a single arm of an antibody, (v) a dAb fragment (Ward et al.,(1989) Nature 341:544-546), which consists of a VH domain; and (vi) anisolated complementarity determining region (CDR). Furthermore, althoughthe two domains of the Fv fragment, VL and VH, are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe VL and VH regions pair to form monovalent molecules (known as singlechain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426; andHuston et al (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Suchsingle chain antibodies are also encompassed within the term“antigen-binding fragment” of an antibody. These antibody fragments areobtained using conventional techniques (including immunization, phagedisplay, and CDR grafting) known to those with skill in the art, and thefragments are screened for utility in the same manner as are intactantibodies.

[0198] An “isolated composition” refers to a composition that is removedfrom at least 90% of at least one component of a natural sample fromwhich the isolated composition can be obtained.

[0199] The invention includes sequences and variants that include one ormore substitutions, e.g., between one and six substitutions. Whether ornot a particular substitution will be tolerated, i.e., will notadversely affect desired biological properties, such as binding activitycan be determined as described in Bowie, et al. (1990) Science247:1306-1310. One or more or all substitutions may be conservative. A“conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Still othersubstitutions may insert a non-naturally occurring amino acid.

[0200] All patent applications, patents, and references cited herein areincorporated by reference in their entirety. Accordingly, U.S.provisional applications Ser. No. 60/331,352 filed Mar. 9, 2001, Ser.No. 60/292,975 filed May 23, 2001, Ser. No. 60/284,534, filed Apr.18,2001, Ser. No. 10/094,401, filed Mar. 8, 2002, and Ser. No. 10/125,869,filed Apr. 18, 2002, U.S. Published application 2003/0069395 areincorporated by reference for all purposes in their entirety.

BRIEF DESCRIPTION OF THE DRAWINGS

[0201]FIG. 1 is an image of a two-dimensional gel of a proteins in afraction of affinity-purified serum albumin and associated proteins.

DETAILED DESCRIPTION

[0202] Serum proteins are important components of the circulatory systemand have wide spread function in physiology and the immune response,among other roles. Characterization of compounds associated with serumproteins provide useful indicia for studying, diagnosing, and monitoringa subject. Serum proteins can be isolated using an affinity reagent.Compounds covalently or non-covalently associated the isolated serumproteins are analyzed, e.g., to determine their identity and/orabundance.

[0203] Isolating Serum Albumin and Associated Compounds

[0204] In one implementation, human serum albumin (HSA) is isolated froma sample, e.g., blood, plasma, or serum. Compounds associated with theserum albumin are then analyzed.

[0205] 1. In a first example, a serum sample is applied to an insolublematrix that includes one or more affinity ligands for HSA. Examples ofaffinity ligands include peptide ligands described below. The matrix iswashed with a physiological-strength buffer (e.g., phosphate bufferedsaline), or more stringent buffers (e.g., including higher ionicstrengths, detergents, chaotropes, and the like).

[0206] HSA and any compounds associated with it are eluted from thematrix, and recovered. Elution can be achieved, for example, by applyingan appropriate buffer that favors the dissociation of HSA from theaffinity ligand or by separating the affinity ligand from the insolublematrix.

[0207] In the eluted material, serum albumin may represent a substantialfraction of the purified composition (see, e.g., FIG. 1 and Example 1).Hence, the associated compounds may constitute less than 10% of thesample. At least some of the associated compounds may be proteinaceous,e.g., peptides, polypeptides, or protein complexes. For example, in thecase of protein complexes, at least some of the associated compounds maybe indirectly associated with HSA. Other compounds may be metabolites,small molecules (e.g., having a molecular weight of less than 5000 or1000 Daltons),

[0208] 2. In a second example, a sample is applied to an insolublematrix that includes one or more affinity ligands for HSA. The matrix iswashed with a physiological-strength buffer (e.g., phosphate bufferedsaline), but not more stringent buffers.

[0209] HSA and any compounds associated with it are removed from thematrix by separating the affinity ligands from the matrix (e.g., theaffinity ligands can be attached to the matrix by a covalent bond thatis cleaved).

[0210] This preparation is then applied to an insoluble matrix thatincludes a thiol-reactive group, e.g., an activated maleimide oriodoacetamide (see also “Thiol Reactive Compounds,” below). Theactivated maleimide, for example, reacts with the free cysteine of HSA,cysteine 34. Few other abundant proteins include a free cysteine whenisolated from serum or from an oxidized sample. After reaction, thematrix is washed to remove proteins and other compounds that are notassociated with HSA.

[0211] Compounds associated with HSA can be released from the matrix byone or more of following processes:

[0212] (a) HSA can be denatured with a chaotrope or other denaturingconditions. The denatured HSA remains covalently bound to the matrix,while non-covalently associated compounds are released from the matrix.Denaturants can be applied to the matrix incrementally, e.g., in a stepor continuous gradient.

[0213] Examples of chaotropes include guanidinium HCI (e.g., >4, 5, or6M) or urea (e.g., >6 or 8M). Examples of other denaturing conditionsinclude acid (e.g., phosphoric acid, pH 1), ionic detergents (e.g., 1%SDS, or greater), heat (e.g., >60° C.) boiling or an organic solvent(e.g., acetonitrile).

[0214] (b) Associated compounds can be eluted by competition using anaffinity ligand that binds to HSA (e.g., an antibody, a peptide ligand,or a compound known to bind HSA, e.g., a long chain fatty acid, a drug,e.g., a drug listed in Table 1, or an endogenous compound, e.g., anendogenous compound listed in Table 2). This process may specificallyelute compounds that associate with a particular epitope of HSA.

[0215] (c) Associated compounds can be separated from each other byselective elution, e.g., using a step or gradient elution, in which asolution parameter is altered (e.g., ionic strength, pH, chaotropeconcentration). Fractions can be collected, and individually analyzed.This process, for example, can used to obtain preparations that includea subset of the associated compounds.

[0216] Fractions of eluted associated compounds can be subjected toadditional purification steps, e.g., a preparative or analytic processdescribed in Scopes (1994) Protein Purification: Principles andPractice, New York: Springer-Verlag.

[0217] The methods described herein can also be used to isolate serumalbumins from other species, e.g., a non-human mammalian species andnon-mammalian species.

[0218] Peptide Ligands that Bind Serum Albumin.

[0219] In some embodiments, peptide ligands are used to isolate a serumalbumin and associated proteins. Provisional patent applications Ser.No. 60/331,352 filed Mar. 9, 2001 and Ser. No. 60/292,975 filed May 23,2001 describe a number of exemplary peptide ligands that bind to serumalbumin. Some exemplary peptide ligands include DX-321, DX-321-A,DX-321-B, DX-236, DX-236-A, and DX-236B.

[0220] DX-321 includes the peptide sequence:

[0221] AEGTGDRNMCKFSWIRSPAFCARADPE(SEQ ID NO: 40). DX-321 binds to humanserum albumin and is useful for isolating human serum albumin andassociated compounds.

[0222] DX-321-A includes the peptide sequence:

[0223] RNMCKFSWIRSPAFCARA (SEQ ID NO: 215).

[0224] DX-321-B includes the peptide sequence:

[0225] CKFSWIRSPAFC (SEQ ID NO: 120)

[0226] DX-236 includes the peptide sequence:

[0227] AEGTGDFWFCDRIAWYPQHLCEFLDPEGGGK(SEQ ID NO: 19). DX-236 binds atleast to a number of mammalian serum albumins and is useful underappropriate conditions as a serum albumin-binding agent that binds toserum albumins from multiple species.

[0228] DX-236A includes the peptide sequence:

[0229] FWFCDRIAWYPQHLCEFLD (SEQ ID NO: 210)

[0230] DX-236B includes the peptide sequence:

[0231] CDRIAWYPQHLC (SEQ ID NO: 9)

[0232] In one implementation, an affinity matrix for purifying serumalbumin includes a plurality of binding ligands, e.g., binding ligandshaving specificity for different epitopes on the serum albumin. Forexample, an affinity matrix for binding HSA can include two differentspecies of HSA binding peptides, e.g., DX-236 and DX-321.

[0233] In addition to peptide ligands, larger polypeptide ligands can beused, e.g., protein that include at least one immunoglobulin domain,e.g., an antibody or antibody fragment.

[0234] Exemplary Serum Albumin Associated Compounds

[0235] A number of endogenous and exogenous compounds are known toassociated with serum albumin. A method described herein can includedetermining whether one or more of such compounds (e.g., a compound inTable 1 or Table 2) is associated with an isolated serum albumin.Compounds other than serum albumin can also be evaluated. TABLE 1 Drugsthat bind to Serum Albumin Salicylate Sulfisoxazole WarfarinPhenylbutazone Digitoxin Indomethacin Tolbutamide Furosenmide PhenytoinChlorpropamide Chlorthiazide Oxacillin Benzylpenicilliln AcetotrizoatePhenol Red Bromscesol green Bromophenol Blue IophenoxateSulfobromophthalein Methyl organge Methyl Red Evans blue Diazepam (S)Ibuprofen Naproxen Octanoate Chlofibrate Chlorpromazine ImipramineQuinidine

[0236] TABLE 2 Endogenous compounds that bind Serum Albumin Long-chainfatty acids Eicosanoids Bile acids Steroids Cortisol ProgesteroneTestosterone Aldosterone Hematin Bilirubin L-Thyroxine L-Tryptophan25-OH-Vitamin D₃ 1,25-(OH)₂-Vitamin D₃ Aquocobalamin Folate AscorbateCopper(II) Zinc(II) Calcium Magnesium Chloride

[0237] Thiol Reactive Compounds

[0238] As described above, thiol reactive groups can be used toimmobilize a serum protein that includes a free cysteine. In particular,serum albumin is an abundant serum protein that includes a freecysteine. For example, cysteine 34 of HSA is typically available forcoupling. Exemplary thiol reactive groups include the following.

[0239] Halogen derivatives. Haloacetyl compounds and benzyl halides,particularly iodo and bromo derivatives, can be reacted with cysteines.For example, iodoacetate can be used to react with cysteines. Thereaction is more specific if the iodoacetate is present in limitingquantities related to the number of available sulfhydryl and underalkaline pH.

[0240] Maleimides. The double bond of maleimides (maleic acid imides)can undergo an alkylation reaction with sulfhydryl groups, resulting ina thioether bond. Maleimides are particularly specific for sulfhydrylsbetween pH 6.5 and 7.5.

[0241] Thiol-Disulfide Exchange Reagents. Cysteines can also becrosslinked using compounds that have a disulfide bond and undergodisulfide exchange with the free cysteine on the serum protein. Pyridyldisulfides, for example, can be generated by reaction of 2-iminothiolanewith 4,4′ dipyridyl disulfide.

[0242] Still other thiol reactive compounds include aziridines, acryloylderivaties, and arylating reagents (such as 2,4 dinitrofluorobenzye).See also Hermanson (1996) “Section 2: Thiol-Reactive Chemical Reactions”of Bioconjugate Techniques Academic Press.

[0243] Peptide Ligands that Bind Immunoglobulins

[0244] In another implementation, a soluble immunoglobulin is isolatedfrom a sample, e.g., blood, plasma, or serum. Compounds associated withthe immunoglobulin are then analyzed.

[0245] A peptide ligand can be used to isolate the solubleimmunoglobulin. WO 2002/086070 and provisional patent application60/284,534, filed Apr. 18, 2001, describe a number of exemplary peptideligands that bind to the Fc region of an immunoglobulin. For example:

[0246] DX249, GDDHMCVYTTWGELIWCDNHEPGPEGGGK (SEQ ID NO: 368) whichexhibits dissociation constants (K_(D)) for human IgG1 of less than 0.1μM at pH 5.7 and less than 0.5 μM at pH 7.4, the segment DX249-A,DHMCVYTTWGELIWCDNH (SEQ ID NO: 260), or the segment DX-249-B,CVYTTWGELIWC (SEQ ID NO: 427);

[0247] DX253, GDRRACSRDWSGALVWCAGHEPGPEGGGK (SEQ ID NO: 369), exhibitsquantitative binding of Fc protein (capture efficiency >90% of totalload), the segment DX253-A, RRACSRDWSGALVWCAGH (SEQ ID NO: 238), or thesegment DX253-B, CSRDWSGALVWC (SEQ ID NO: 428);

[0248] DX398, AGAATCSTSYWYYQWFCTDSPGPEGGGK (SEQ ID NO: 375), DX398-A:AATCSTSYWYYQWFCTDS (SEQ ID NO: 348) or DX398-B: CSTSYWYYQWFC (SEQ ID NO:429);

[0249] DX252, GDDDHCYWFREWFNSECPHGEPGPEGGGK (SEQ ID NO: 378), exhibitsdissociation constants (K_(D)) for human IgG3 of less than 0.1 μM at pH5.7 and in the range of ˜2.1 μM to ˜3.4 μM for IgG1, IgG2, IgG3, andIgG4 at pH 7.4; and

[0250] DX254, AGYYWCNYWGLCPDQGTPGPEGGGK (SEQ ID NO: 379), exhibitsdissociation constants (K_(D)) for human IgG1 of less than 0.1 μM at pH5.7, less than 2.0 μM at pH 7.4, and less than 1.0 μM at pH 9.3.

[0251] In one implementation, a sample is contacted to an affinitymatrix that includes ligands that bind to immunoglobulins.Immunoglobulin and associated compounds are isolated. In one example,the isolated material is analyzed, e.g., to characterize antigensassociated with immunoglobulin. In another example, the isolatedmaterial is cultured, e.g., to identify a pathogen bound by theimmunoglobulin.

[0252] Analyzing Associated Compounds

[0253] A fraction of a serum protein and associated compounds can beanalyzed by a number of processes. Exemplary methods for analyzingproteinaceous compounds associated with a serum protein include: sodiumdodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 2-D gelelectrophoresis (iso-electric focusing and PAGE), HPLC, FPLC(ion-chromatography, size exclusion chromatography, hydrophobicinteraction chromatography, and the like), immuno-analysis (e.g.,immuno-blots, enzyme-linke.d immunosorbent assay (ELISA),immunoprecipitation, and the like), mass spectroscopy, and proteinsequencing.

[0254] Exemplary methods for analyzing a non-proteinaceous compoundassociated with a serum protein include: mass spectroscopy (e.g.,GC-mass spec or “GC/MS”), thin-layer chromatography, and other chemicaldetection methods.

[0255] 1D gels. SDS-PAGE can be used to separate proteins by theirapparent/approximate molecular weight in an acrylamide gel. Theconcentration of acrylamide can be varied according to the expected sizeor a gradient of acrylamide concentration can be used. Afterelectrophoresis, proteins can be stained using a variety of dyes,include Coomassie Blue, silver stains, and fluorescent dyes such asSypro Red (Molecular Probes, Inc., Eugene, Oreg.). The acrylamide gelcan be imaged, e.g., to determine relative concentration of resolvedbands of proteins. The image can be stored in a computer database.

[0256] 2D gels. This method can be used to separate polypeptidesaccording to two properties, isoelectric point (pI) and apparentmolecular weight (MW). Proteins are first separated by PI (isoelectricfocusing) and then separated according to apparent molecular weight bySDS-PAGE. The 2D gel is stained and imaged. The detected “spots” ofproteins provide information about the identity, modification state, andrelative abundance of each protein. Proteins can also be excised fromspots and further characterized by mass spectroscopy or proteinsequencing.

[0257] Isoelectric focusing for the first dimension can utilizeimmobilized pH gradient strips, e.g., in which polycarboxylic acidampholytes are immobilized. Strips can be produced that focus within adesired pH range, both narrow and wide. A strip can be selected for theappropriate degree of resolution.

[0258] After isoelectric focusing, protein in the strip are denaturedand reduced, e.g., using SDS and a thiol reductant. The strip isattached to an SDS-PAGE slab gel and proteins are separated by molecularweight.

[0259] Immuno-Detection. Antibodies, either characterized oruncharacterized, can be used to identify compounds associated with aserum protein. The antibodies can be applied to a separated sample(e.g., an electrophoresed sample, as in a Western blot) or to the sampleas a whole (e.g., an ELISA). An antibody can also be used toimmunoprecipitate the compound. The antibody can be coupled to a labelor signal generator to enable detection of the compound. Antibodyfragments and other derivatives can also be used.

[0260] Mass Spectroscopy. Time-of flight mass spectrometry (TOF-MS) andelectrospray mass spectrometry can be used to characterize compoundsassociated with a serum protein. TOF-MS is sensitive, highly accurate,and rapid (R. J. Cotter, (1992) Anal. Chem. 64:1027. For example, TOF-MScan record a complete mass spectrum on a microsecond timescale.

[0261] One common TOF-MS method is matrix-assisted laserdesorption/ionization (MALDI) (Karas and Hillenkamp, Anal. Chem. 60,2299 (1988). This method is amenable to the mass spectrometry tooligonucleotides and nucleic acids. See generally, P. Limbach et al,“Characterization of oligonucleotides and nucleic acids by massspectrometry”, In Current Opinion in Biotechnology, 6, 96-102 (1995).

[0262] Mass spectroscopy can be combined with protease digestion todetermine the precise molecular weight of proteolytic fragments of aprotein. This information can be compared to a computer sequencedatabase to infer the sequence of the protein. For example, the databasecan includes predicted protein sequences from genome sequence. See,e.g., Zhang and Chait (2000) Anal. Chem. 72:2482. Mass spectroscopy canalso be used to determine the modification state of a protein (e.g.,oxidation, glycosylation).

[0263] Exemplary proteases for mapping proteolytic fragments include:elastase, trypsin, chymotrypsin, pepsin, papain, and Glu-C. Certainchemical agents can also be used, e.g., formic acid and cyanogenbromide.

[0264] For matrix-assisted laser desorption/ionization mass spectrometry(MALDI-MS or MALDI-TOF), a proteolyzed sample is combined with a matrix(e.g., α-cyano-4-hydroxycinnamic acid), sinapinic acid, or gentisicacid), and dried on a mass spectroscopy plate. The plate is then placedin a mass spectrometer where the protein fragments are ionized, and thenanalyzed for their time of flight. Accurate molecular weights aredetermined from these measurements.

[0265] Protein Sequencing. The N-termini of a purified protein (e.g., 2to 5 picomoles of the protein) can be sequenced by Edman degradation.This process can be automated. N-terminal sequence information can becombined with mass spectroscopy information and comprehensive databasesto unambiguously identify a protein.

[0266] Peptide and Protein Arrays

[0267] In some implementations, an array of proteins and/or peptideligands, at least some of which bind to serum proteins can be used. Forexample, the array can include one or more peptide ligands describedherein. In a particular example, the array includes at least twodifferent peptide ligands that bind to serum albumin.

[0268] In general, a sample is contacted to the array, and complexeswithin the sample are allowed to bind to ligands on the array, e.g., sothat different complexes of a serum binding protein are isolated by thedifferent ligands. Each discrete address can be evaluated separately.

[0269] Methods of producing polypeptide arrays are described, e.g., inDe Wildt et al. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal. Biochem. 270:103- 111; Ge (2000) Nucleic Acids Res. 28, e3,I-VII; MacBeath and Schreiber (2000) Science 289:1760-1763; U.S. Pat.No. 2002-192,673 WO 01/98534, WO01/83827, WO02/12893, WO 00/63701, WO01/40803 and WO 99/51773. In some implementations, polypeptides(including peptides) are spotted onto discrete addresses of the array,e.g., at high speed, e.g., using commercially available roboticapparati, e.g., from Genetic MicroSystems or BioRobotics. The arraysubstrate can be, for example, nitrocellulose, plastic, glass, e.g.,surface-modified glass. The array can also include a porous matrix,e.g., acrylamide, agarose, or another polymer.

[0270] Arrays of peptides can be similarly produced. In addition,peptides can be directly synthesized in an array format, e.g., accordingto U.S. Pat. No. 5,143,854. It may also be possible to use nucleic acidaptamers as ligands to isolate serum proteins and associated complexes.

[0271] Analysis of Altered or Abnormal States

[0272] The methods described herein can be used to characterize analtered or abnormal state of a subject. For example, a profile ofcompounds associated with one or more serum proteins can be determinedfor the subject at one or more instances. The subject can be a diseasedsubject, a genetically altered subject, a subject afflicted by a geneticdisorder, a subject exposed to toxins (e.g., environmental toxins,narcotics, and so forth), or a subject receiving a treatment. Forinstance, in the case of monitoring a treatment of a subject, theprofile can be determined prior to treatment, and at regular intervalsafter treatment. The profile may provide information about the pathologyof the subject (e.g., if diseased), the abundance of an administereddrug, or a drug by-product in the subject's serum, or the abundance ofnatural components whose levels might be affected by the treatment.

[0273] Drug Testing

[0274] The methods described herein can also be used to test theaffinity of a test compound, e.g., a drug, for serum components.Information about whether a potential pharmaceutical interacts with aserum protein is useful for characterizing its efficacy and utility as atherapeutic agent.

[0275] For example, the test compound can be mixed with a biologicalsample, such as blood or serum or an at least partially purifiedpreparation of a serum protein (e.g., a recombinant serum protein).After incubation, one or more serum proteins is isolated from thesample, e.g., using an affinity reagent, e.g., a reagent that includes apeptide ligand described herein. Binding of the test compound to theisolated serum protein can be determined, e.g., by quantifying theamount of test compound that is isolated with the serum protein. Thetest compound can be unlabeled or labeled (e.g., using a radioactivelabel or fluorescent label). A labeled test compound can be directlydetected, e.g., using a scintillation proximity assay or fluorescenceassay.

[0276] Unlabeled compounds can also be detected. For example, massspectrometry can be used for detecting with unlabeled compounds. Inanother example, unlabeled compounds can be detected in a competitionassay. Unlabeled compounds bound to the serum protein are separated fromthe serum protein and added to a binding reaction, e.g., in a well of amicrotitre plate. The binding reaction includes an antibody that bindsto the unlabeled compound and a known quantity of labeled compound. Theamount of unlabeled compound present is determined by measuring theamount of labeled compound bound by the antibody. Competition by theunlabeled compound reduces the amount of bound labeled compound.

[0277] A related method includes administering the test compound to asubject, e.g., an animal model. After one or more appropriate intervals,a blood or serum sample is extracted from the subject. The amount oftest compound associated with a serum protein can be determined, e.g.,as described above. If the subject is a non-human mammal, an affinityligand that is not species specific can be used.

[0278] Compounds that Modulate Interactions with a Serum Protein

[0279] It is also possible to screen for modulator compounds thatmodulate the interaction of a serum-protein binding compound and a serumprotein, e.g., serum albumin. For example, it is possible to use a highthroughput screen for compounds that disrupt (or enhance) theinteraction between a naturally-occurring protein and serum protein.

[0280] One method for screening includes: contacting a candidatemodulator compound to a complex that includes the serum protein-bindingcompound and the serum protein; and evaluating the interaction betweenthe serum protein-binding compound and the serum protein. In oneimplementation, the serum protein is bound to an affinity reagent (e.g.,a peptide ligand) and isolated. The isolated material is analyzed todetermine the presence and/or amount of the serum protein-bindingcompound. A modulator compound that disrupts the interaction between theserum protein-binding compound and the serum protein may reduce orprevent isolation of the serum protein-binding compound.

[0281] A related method for screening involves contacting the serumprotein to the candidate modulator compound, and subsequently adding theserum protein-binding compound to determine if the candidate modulatorimpairs or enhances the interaction between the serum protein and theserum protein-binding compound. Likewise, all three components can becombined together and then analyzed.

[0282] Identifying Binding Ligands for Serum Proteins.

[0283] Ligands that bind to a serum protein can be identified by avariety of methods including screening a display library. For example,phage display can be used to screen a library of linear or cyclicpeptides for peptides that bind to a given serum protein. In addition,ligands that include an immunoglobulin domain, e.g., antibodies, can begenerated (e.g., by immunization, or display library screening).

[0284] Peptide ligands that bind to human serum albumin and the Fcregion of immunoglobulin are described herein and in U.S. provisionalapplications Ser. No. 60/331,352 filed Mar. 9, 2001, Ser. No. 60/292,975filed May 23, 2001, Ser. No. 60/284,534, filed Apr. 18, 2001. Similarly,ligands can be isolated that bind to a serum protein such as:transferrin, α macroglobulins, ferritin, apolipoproteins, transthyretin,a protease inhibitor found in serum, retinol binding protein,thiostatin, α-fetoprotein, vitamin-D binding protein, or afamin.

[0285] One method of identifying a binding ligand for a serum protein isto screen a display library. A display library is a collection ofentities; each entity includes an accessible polypeptide component and arecoverable component that encodes or identifies the polypeptidecomponent. The polypeptide component can be of any length, e.g. fromthree amino acids to over 300 amino acids. In a selection, thepolypeptide component of each member of the library is probed with theserum protein and if the polypeptide component binds to the protein, thedisplay library member is identified, typically by retention on asupport.

[0286] The screening of display libraries is advantageous, in that verylarge numbers (e.g., 5×10⁹) of potential binders can be tested, andsuccessful binders isolated in a short period of time. Further, unlikeimmunization, ligands can be identified that bind to epitopes of serumproteins that are conserved among different species.

[0287] Retained display library members are recovered from the supportand analyzed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the polypeptide component andpurification of the polypeptide component for detailed characterization.

[0288] A variety of formats can be used for display libraries. Examplesinclude the following.

[0289] Phage Display. One format utilizes viruses, particularlybacteriophages. This format is termed “phage display.” The polypeptidecomponent is typically covalently linked to a bacteriophage coatprotein. The linkage results form translation of a nucleic acid encodingthe polypeptide component fused to the coat protein. The linkage caninclude a flexible peptide linker, a protease site, or an amino acidincorporated as a result of suppression of a stop codon. Phage displayis described, for example, in Ladner et al., U.S. Pat. No. 5,223,409;Smith (1985) Science 228:1315-1317; WO 92/18619; WO 91/17271; WO92/20791; WO 92/15679; WO 93/01288; WO 92/01047; WO 92/09690; WO90/02809; de Haard et al. (1999) J. Biol. Chem 274:18218-30; Hoogenboomet al. (1998) Immunotechnology 4:1-20; Hoogenboom et al. (2000) ImmunolToday 2:371-8; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum Antibod Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al.(1992) J Mol Biol 226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)Bio/Technology 9:1373-1377; Rebar et al. (1996) Methods Enzymol.267:129-49; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; andBarbas et al. (1991) PNAS 88:7978-7982.

[0290] Phage display systems have been developed for filamentous phage(phage fl, fd, and M13) as well as other bacteriophage (e.g. T7bacteriophage and lambdoid phages; see, e.g., Santini (1998) J. MolBiol. 282:125-135; Rosenberg et al. (1996) Innovations 6:1-6; Houshmetal. (1999) Anal Biochem 268:363-370). The filamentous phage displaysystems typically use fusions to a minor coat protein, such as gene IIIprotein, and gene VIII protein, a major coat protein, but fusions toother coat proteins such as gene VI protein, gene VII protein, gene IXprotein, or domains thereof can also been used (see, e.g., WO 00/71694).In a preferred embodiment, the fusion is to a domain of the gene IIIprotein, e.g., the anchor domain or “stump,” (see, e.g., U.S. Pat. No.5,658,727 for a description of the gene III protein anchor domain).

[0291] The valency of the polypeptide component can also be controlled.Cloning of the sequence encoding the polypeptide component into thecomplete phage genome results in multivariant display since allreplicates of the gene III protein are fused to the polypeptidecomponent. For reduced valency, a phagemid system can be utilized. Inthis system, the nucleic acid encoding the polypeptide component fusedto gene III is provided on a plasmid, typically of length less than 700nucleotides. The plasmid includes a phage origin of replication so thatthe plasmid is incorporated into bacteriophage particles when bacterialcells bearing the plasmid are infected with helper phage, e.g. M13K01.The helper phage provides an intact copy of gene III and other phagegenes required for phage replication and assembly. The helper phage hasa defective origin such that the helper phage genome is not efficientlyincorporated into phage particles relative to the plasmid that has awild type origin.

[0292] Bacteriophage displaying the polypeptide component can be grownand harvested using standard phage preparatory methods, e.g. PEGprecipitation from growth media.

[0293] After selection of individual display phages, the nucleic acidencoding the selected polypeptide components, by infecting cells usingthe selected phages. Individual colonies or plaques can be picked, thenucleic acid isolated and sequenced.

[0294] Cell-based Display. In still another format the library is acell-display library. Proteins are displayed on the surface of a cell,e.g., a eukaryotic or prokaryotic cell. Exemplary prokaryotic cellsinclude E. coli cells, B. subtilis cells, spores (see, e.g., Lu et al.(1995) Biotechnology 13:366). Exemplary eukaryotic cells include yeast(e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe, Hanseula, orPichia pastoris). Yeast surface display is described, e.g., in Boder andWittrup (1997) Nat. Biotechnol. 15:553-557 and WO03029456. Thisapplication describes a yeast display system that can be used to displayimmunoglobulin proteins such as Fab fragments, and the use of mating togenerate combinations of heavy and light chains.

[0295] In one embodiment, variegate nucleic acid sequences are clonedinto a vector for yeast display. The cloning joins the variegatedsequence with a domain (or complete) yeast cell surface protein, e.g.,Aga2, Aga1, Flo1, or Gas1. A domain of these proteins can anchor thepolypeptide encoded by the variegated nucleic acid sequence by atransmembrane domain (e.g., Flo1) or by covalent linkage to thephospholipid bilayer (e.g., Gas1). The vector can be configured toexpress two polypeptide chains on the cell surface such that one of thechains is linked to the yeast cell surface protein. For example, the twochains can be immunoglobulin chains.

[0296] Ribosome Display. RNA and the polypeptide encoded by the RNA canbe physically associated by stabilizing ribosomes that are translatingthe RNA and have the nascent polypeptide still attached. Typically, highdivalent Mg²⁺ concentrations and low temperature are used. See, e.g.,Mattheakis et al. (1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes etal (2000) Nat Biotechnol. 18:1287-92; Hanes et al. (2000) MethodsEnzymol. 328:404-30. and Schaffitzel et al. (1999) J Immunol Methods.231(1-2):119-35.

[0297] Peptide-Nucleic Acid Fusions. Another format utilizespeptide-nucleic acid fusions. Polypeptide-nucleic acid fusions can begenerated by the in vitro translation of mRNA that include a covalentlyattached puromycin group, e.g., as described in Roberts and Szostak(1997) Proc. Natl. Acad. Sci. USA 94:12297-12302, and U.S. Pat. No.6,207,446. The mRNA can then be reverse transcribed into DNA andcrosslinked to the polypeptide.

[0298] Other Display Formats. Yet another display format is anon-biological display in which the polypeptide component is attached toa non-nucleic acid tag that identifies the polypeptide. For example, thetag can be a chemical tag attached to a bead that displays thepolypeptide or a radiofrequency tag (see, e.g., U.S. Pat. No.5,874,214).

[0299] Scaffolds. Scaffolds for display can include: antibodies (e.g.,Fab fragments, single chain Fv molecules (scFV), single domainantibodies, camelid antibodies, and camelized antibodies); T-cellreceptors; MHC proteins; extracellular domains (e.g., fibronectin TypeIII repeats, EGF repeats); protease inhibitors (e.g., Kunitz domains,ecotin, BPTI, and so forth); TPR repeats; trifoil structures; zincfinger domains; DNA-binding proteins; particularly monomeric DNA bindingproteins; RNA binding proteins; enzymes, e.g., proteases (particularlyinactivated proteases), RNase; chaperones, e.g., thioredoxin, and heatshock proteins; and intracellular signaling domains (such as SH2 and SH3domains).

[0300] Another useful type of scaffolding domain is the immunoglobulin(Ig) domain. Methods using immunoglobulin domains for display are alsoknown (see, e.g., Haard et al. (1999) J. Biol. Chem 274:18218-30;Hoogenboom et al. (1998) Immunotechnology 4:1-20. and Hoogenboom et al.(2000) Immunol Today 21:371-8).

[0301] Synthetic Peptides. The binding ligand can include a syntheticpeptide, e.g., an artificial peptide of 30 amino acids or less. Thesynthetic peptide can include one or more disulfide bonds. Othersynthetic peptides, so-called “linear peptides,” are devoid ofcysteines. Synthetic peptides may have little or no structure insolution (e.g., unstructured), heterogeneous structures (e.g.,alternative conformations or “loosely structured), or a singular nativestructure (e.g., cooperatively folded). Some synthetic peptides adopt aparticular structure when bound to a target molecule. Some exemplarysynthetic peptides are so-called “cyclic peptides” that have at leastdisulfide bond, and, for example, a loop of about 4 to 12 non-cysteineresidues.

[0302] Peptide sequences that bind a molecular target are selected froma phage-display library. After identification, such peptides can beproduced synthetically or by recombinant means. The sequences can beincorporated (e.g., inserted, appended, or attached) into longersequences.

[0303] Exemplary Phage Display Libraries for Identifying BindingPeptides

[0304] Display libraries exhibiting variegated heterologous peptides onthe surface of recombinant phage or other genetic packages (bacteria,yeast, other host cells) may be prepared in any of several ways known inthe art. See, e.g., Kay et al., Phage Display of Peptides and Proteins:A Laboratory Manual (Academic Press, Inc., San Diego 1996) and U.S. Pat.No. 5,223,409 (Ladner et al.).

[0305] The following are six exemplary phage libraries that can bescreened to find at least some of the polypeptide ligands describedherein. Each library displays a short, variegated exogenous peptide onthe surface of M13 phage. The peptide display of five of the librarieswas based on a parental domain having a segment of 4, 5, 6, 7, 8, or 10amino acids, respectively, flanked by cysteine residues. The pairs ofcysteines are believed to form stable disulfide bonds, yielding a cyclicdisplay peptide. The cyclic peptides are displayed at the amino terminusof protein III on the surface of the phage. The libraries weredesignated TN6/6, TN8/9, TN9/4, TN10/9, and TN12/1. A phage library witha 20-amino acid linear display was also screened; this library wasdesignated Lin20.

[0306] The TN6/6 library was constructed to display a single cyclicpeptide contained in a 12-amino acid variegated template. The TN6/6library utilized a template sequence ofXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Cys₉-Xaa₁₀-Xaa₁₁-Xaa₁₂ (SEQ IDNO: 21), where each variable amino acid position in the amino acidsequence of the template is indicated by a subscript integer. Eachvariable amino acid position (Xaa) in the template was varied,independently, to permit the following substitutions: residues Xaa₁ andXaa₁₂ were varied to contain any of the following 14 amino acids: Ala,Asp, Phe, Gly, His, Leu, Asn, Pro, Gln, Arg, Ser, Val, Trp, and Tyr; andresidues Xaa₂, Xaa₃ Xaa₅, Xaa₆, Xaa₇, Xaa₈, Xaa₁₀, and Xaa₁₁ wereindependently varied to contain any of the common α-amino acids, exceptcysteine (Cys). The number of potential designed sequences is 3.3×10¹²;2.0×10⁸ independent transformants were included in the library.

[0307] The TN8/9 library was constructed to display a single bindingloop contained in a 14-amino acid template. The TN8/9 library utilized atemplate sequence ofXaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Cys-Xaa₁₂-Xaa₁₃-Xaa₁₄(SEQ ID NO: 25). The amino acids at position 1, 2, 3, 5, 6, 7, 8, 9, 10,12, 13, and 14 in the template were varied to permit any amino acidexcept cysteine (Cys).

[0308] The TN9/4 library was constructed to display a single bindingloop contained in a 15-amino acid template. The TN9/4 library utilized atemplate sequence Xaa₁-Xaa₂-Xaa₃-Cys-Xaa₅-Xaa₆-Xaa₇-Xaa₈Xaa₉-Xaa₁₀-Xaa₁₁-Cys-Xaa₁₃-Xaa₁₄-Xaa₁₅ (SEQ ID NO: 424 The amino acidsat position 1, 2, 3, 5, 6, 7, 8, 9, 10, 11, 13, 14 and 15 in thetemplate were varied to permit any amino acid except cysteine (Cys).

[0309] The TN10/9 library was constructed to display a single cyclicpeptide contained in a 16-amino acid variegated template. The TN10/9library utilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₂-Cys₁₃-Xaa₁₄-Xaa₁₅-Xaa₁₆(SEQ ID NO: 22), where each variable amino acid position in the aminoacid sequence of the template is indicated by a subscript integer. Eachvariable amino acid position (Xaa) was varied independently to permitthe following substitutions. The amino acid positions Xaa₁, Xaa₂, Xaa₁₅and Xaa₁₆ of the template were varied, independently, to permit each ofthe amino acids selected from a group of ten amino acids consisting ofAsp, Phe, His, Leu, Asn, Pro, Arg, Ser, Trp, and Tyr; the amino acids atamino acid positions Xaa₃ and Xaa₁₄ in the template were varied,independently, to permit each amino acid selected from the group offourteen amino acids consisting of Ala, Asp, Glu, Phe, Gly, His, Leu,Asn, Pro, Arg, Ser, Val, Trp, and Tyr; the amino acids at amino acidpositions Xaa₅, Xaa_(6,) Xaa₇, Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁ and Xaa₁₂ (i.e.,between the invariant cysteine residues at positions 4 and 13 in thetemplate) were varied, independently, to permit each of the commonα-amino acids, except cysteine. The number of potential designedsequences is 3.0×10¹⁶; and about 2.5×10⁸ independent transformants wereincluded in the library.

[0310] The TN12/1 library was constructed to display a single cyclicpeptide contained in an 18-amino acid template. The TN12/1 libraryutilized a template sequenceXaa₁-Xaa₂-Xaa₃-Cys₄-Xaa₅-Xaa₆-Xaa₇-Xaa₈-Xaa₉-Xaa₁₀-Xaa₁₁-Xaa₁₂-Xaa₁₃-Xaa₁₄-Cys₁₅-Xaa₁₆-Xaa₁₇-Xaa₁₈(SEQ ID NO: 23), where each variable amino acid position in the aminoacid sequence of the template is indicated by a subscript integer. Theamino acid positions Xaa₁, Xaa₂, Xaa₁₇ and Xaa₁₈ of the template werevaried, independently, to permit each amino acid selected from the groupof 12 amino acids consisting of Ala, Asp, Phe, Gly, His, Leu, Asn, Pro,Arg, Ser, Trp, and Tyr. The amino acid positions Xaa₃, Xaa₅, Xaa_(6,)Xaa₇, Xaa₈, Xaa₉, Xaa₁₀, Xaa₁₁, Xaa₁₂, Xaa₁₃, Xaa₁₄, Xaa₁₆, of the thetemplate were varied, independently, to permit each of the common(α-amino acids, except cysteine.

[0311] The Lin20 library was constructed to display a single linearpeptide in a 20-amino acid template. The amino acids at each position inthe template were varied to permit any amino acid except cysteine (Cys).

[0312] The techniques discussed in Kay et al., Phage Display of Peptidesand Proteins: A Laboratory Manual (Academic Press, Inc., San Diego 1996)and U.S. Pat. No. 5,223,409 are useful for preparing a library ofpotential binders corresponding to the selected parental template. Thelibraries described above can be prepared according to such techniques,and screened for binding peptides against a serum protein, eitherimmobilized on a solid surface or free in solution.

[0313] Screening Phage Display Libraries for Serum Protein BindingPeptides

[0314] In a typical screen, a phage library is contacted with andallowed to bind the target compound, e.g., a serum protein of interest,or a particular fragment or subcomponent thereof. To facilitateseparation of binders and non-binders in the screening process, it isoften convenient to immobilize the target compound on a solid support,although it is also possible to first permit binding to the targetcompound in solution and then segregate binders from non-binders bycoupling the target compound to a support. By way of illustration, whenincubated in the presence of the target, phage bearing a target-bindingmoiety form a complex with the target compound immobilized on a solidsupport whereas non-binding phage remain in solution and may be washedaway with buffer. Bound phage may then be liberated from the target by anumber of means, such as changing the buffer to a relatively high acidicor basic pH (e.g., pH 2 or pH 10), changing the ionic strength of thebuffer, adding denaturants, or other known means.

[0315] For example to identify HSA-binding ligands, HSA can be adsorbed(by passive immobilization) to a solid surface, such as the plasticsurface of wells in a multi-well assay plate, and then an aliquot of aphage display library was added to a well under appropriate conditionsthat maintain the structure of the immobilized HSA and the phage, suchas pH 6-7. Phage in the libraries that display peptide loop structuresthat bind the immobilized HSA are retained bound to the HSA adhering tothe surface of the well and non-binding phage can be removed. Phagebound to the immobilized HSA may then be eluted by washing with a buffersolution having a relatively strong acid pH (e.g., pH 2) or an alkalinepH (e.g., pH 8-9). The solutions of recovered phage that are eluted fromthe HSA are then neutralized and may, if desired, be pooled as anenriched mixed library population of phage displaying serum albuminbinding peptides. Alternatively the eluted phage from each library maybe kept separate as a library-specific enriched population of HSAbinders. Enriched populations of phage displaying serum albumin bindingpeptides may then be grown up by standard methods for further rounds ofscreening and/or for analysis of peptide displayed on the phage and/orfor sequencing the DNA encoding the displayed binding peptide.

[0316] One of many possible alternative screening protocols uses HSAtarget molecules that are biotinylated and that can be captured bybinding to streptavidin, for example, coated on particles. As isdescribed in an example below, phage displaying HSA binding peptideswere selected from a library in such a protocol in which phagedisplaying HSA binding peptides were bound to acaprylate-biotinylated-HSA in solution at pH 7.4 in phosphate bufferedsaline (PBS) supplemented with 0.1% Tween 20 nonionic detergent and also0.1 % sodium caprylate, which is known to stabilize HSA againsttemperature-induced denaturation and proteolytic attack. Thecaprylate-biotinylated-HSA/phage complexes in solution were thencaptured on streptavidin-coated magnetic beads. Phage were subsequentlyeluted from the beads for further study.

[0317] Recovered phage may then be amplified by infection of bacterialcells, and the screening process may be repeated with the new pool ofphage that is now depleted in non-HSA binders and enriched in HSAbinders. The recovery of even a few binding phage is sufficient to carrythe process to completion. After a few rounds of selection, the genesequences encoding the binding moieties derived from selected phageclones in the binding pool are determined by conventional methods,revealing the peptide sequence that imparts binding affinity of thephage to the target. An increase in the number of phage recovered aftereach round of selection and the recovery of closely related sequencesindicate that the screening is converging on sequences of the libraryhaving a desired characteristic.

[0318] After a set of binding polypeptides is identified, the sequenceinformation may be used to design other, secondary libraries, biased formembers having additional desired properties.

[0319] Display technology can also be used to obtain ligands, e.g.,antibody ligands or peptide ligands, that bind to particular epitopes ofa target. This can be done, for example, by using competing non-targetmolecules that lack the particular epitope or are mutated within theepitope, e.g., with alanine. Such non-target molecules can be used in anegative selection procedure as described below, as competing moleculeswhen binding a display library to the target, or as a pre-elution agent,e.g., to capture in a wash solution dissociating display library membersthat are not specific to the target.

[0320] The binding properties of a ligand that binds a serum protein canbe readily assessed using various assay formats. For example, thebinding property of a ligand can be measured in solution by fluorescenceanisotropy, which provides a convenient and accurate method ofdetermining a dissociation constant (K_(D)) of a binding moiety for aserum albumin from one or more different species. In one such procedure,a binding moiety described herein is labeled with fluorescein. Thefluorescein-labeled binding moiety may then be mixed in wells of amulti-well assay plate with various concentrations of a particularspecies of serum albumin. Fluorescence anisotropy measurements are thencarried out using a fluorescence polarization plate reader. The bindinginteraction between a serum protein and a non-covalently associatedcompound can be similarly characterized. Other solution measures forstudying binding properties include fluorescence resonance energytransfer (FRET) and NMR.

[0321] Binding properties can also be characterized using a methodwherein one binding partner is immobilized. Such methods include ELISAand surface plasmon resonance.

[0322] Serum Binding Protein Ligand Variants

[0323] It is also possible to use a variant of a serum binding proteinligand described herein or isolated by a method described herein. Anumber of variants are possible. A variant can be prepared and thentested, e.g., using a binding assay described above (such asfluorescence anisotropy). If the variant is function, it can be used asan affinity reagent to isolate a serum protein and associated compounds.

[0324] One type of variant is a truncation of a ligand described hereinor isolated by a method described herein. In this example, the variantis prepared by removing one or more amino acid residues of the ligandcan be removed from the N or C terminus. In some cases, a series of suchvariants is prepared and tested. Information from testing the series isused to determine a region of the ligand that is essential for bindingthe serum protein. A series of internal deletions or insertions can besimilarly constructed and tested.

[0325] Another type of variant is a substitution. In one example, theligand is subjected to alanine scanning to identify residues thatcontribute to binding activity. In another example, a library ofsubstitutions at one or more positions is constructed. The library maybe unbiased or, particularly if multiple positions are varied, biasedtowards an original residue.

[0326] A related type of variant is a ligand that includes one or morenon-naturally occurring amino acids. Such variant ligands can beproduced by chemical synthesis. One or more positions can be substitutedwith a non-naturally occurring amino acid. In some cases, thesubstituted amino acid may be chemically related to the originalnaturally occurring residue (e.g., aliphatic, charged, basic, acidic,aromatic, hydrophilic) or an isostere of the original residue.

[0327] It may also be possible to include non-peptide linkages and otherchemical modification. For example, part or all of the ligand may besynthesized as a peptidomimetic, e.g., a peptoid (see, e.g., Simon etal. (1992) Proc. Natl. Acad. Sci. USA 89:9367-71 and Horwell (1995)Trends Biotechnol.13:132-4)

[0328] For example, variants of serum albumin-binding ligands (such asDX-321, DX-321-A, DX-321-B, DX-36, DX-236-A, and DX-236B) andimmunoglobulin-binding ligands (such as DX249, DX249-A, DX253, DX-253-1,DX398, and DX398-A) can be used.

[0329] Sequences of Human Serum Proteins

[0330] The amino acid sequences of human serum proteins are well knownand can be found in public sequence repositories, e.g., GenBank(National Center for Biotechnology Information, National Institutes ofHealth, Bethesda Md.). Further, in the human population, natural geneticvariation can result in amino acid differences between serum proteinsamong individuals.

[0331] The following sequences are examples of at least some human serumprotein amino acid sequences from particular individuals.

[0332] In many individuals, HSA has the amino acid sequence listed inSwissProt entry: P02768 and/or the following matureDAHKSEVAHRFKDLGEENFKALVLIAFAQYLQQCPFEDHVKLVNEVTEF (SEQ ID NO:26)AKTCVADESAENCDKSLHTLFGDKLCTVATLRETYGEMADCCAKQEPERNECFLQHKDDNPNLPRLVRPEVDVMCTAYHDNEETFLKKYLYELARRHPYFYAYELLFFAKRYKAAFTECCQAADKAACLLPKLDELRDEGKASSAKQRLKCASLQKFGERAFKAWAVARLSQRFPKAEFAEVSKLVTDLTKVHTECCHGDLLECADDRADLAKYICENQDSISSKLKECCEKPLLEKSHCIAEVENDEMPADLPSLAADFVESKDVCKNYAEAKDVFLGMFLYEYARRHPDYSVVLLLRLAKTYETTLEKCCAAADPHECYAKVFDEFKPLVEEPQNLIKQNCELFEQLGEYKFQNALLVRYTKKVPQVSTPTLVEVSRNLGKVGSKCCKITPEAKRMPCAEDYLSVVLNQLCVLHEKTPVSDRVTKCCTESLVNRRPCFSALEVDETYVPKEFNAETFTFHADICTLSEKERQIKKQTALVELVKHKPKATKEQLKAVMDDFAAFVEKCCKADDKETCFAEEGKKLVAASQAALGL.

[0333] Examples of human serum albumin variants include H27Q, H27Y,E106K, R122S, E378K, E400K, and E529K (numbered using the unprocessedsequence, wherein the initial D of SEQ ID NO: 26 corresponds to residue25 of the unprocessed sequence).

[0334] Automated Methods and Information Management

[0335] Any and all aspects of the serum protein analysis platform can beautomated. Automation, for example, can be used to process multipledifferent samples automatically. Liquid handling units can be used toisolate compounds associated with a serum protein from the sample andcan automatically subject the isolated compounds to analytical methodssuch as electrophoresis and/or mass spectroscopy.

[0336] Equipment. Various robotic devices can be employed in theautomation process. These include multi-well plate conveyance systems,magnetic bead particle processors, and liquid handling units. Thesedevices can be built on custom specifications or purchased fromcommercial sources, such as Autogen (Framingham Mass.), Beckman Coulter(USA), Biorobotics (Woburn Mass.), Genetix (New Milton, Hampshire UK),Hamilton (Reno Nev.), Hudson (Springfield N.J.), Labsystems (Helsinki,Finland), Packard Bioscience (Meriden Conn.), and Tecan (Mannedorf,Switzerland).

[0337] Information Management. Information generated by the platform canbe stored in a computer database (e.g., in digital form). Information,including information that describes the characterization of compoundsassociated with a serum protein, is stored in a central database. Forexample, the database can include information that describes a propertyof an associated compound (e.g., protein sequence, chemical structure,abundance, modification state, etc. and information that describes thesample (e.g., identity of its source, date, processing method,pathology, treatment, etc.). These items of information can beassociated with each other. For example, a query about a particularstate, e.g., a particular disease or treatment, can be used to identifyproperties of associated compounds found in that state. Likewise, aparticular property of one or more associated compounds can be used as aquery to identify states with which the property is prevalent.

[0338] The database can also include a profile, e.g., a description of aplurality of associated compounds from a sample in a particular state.Software can be used to compare profiles, e.g., to evaluate a givensample by comparison to the collection of profiles. One or more similarprofiles can be used to infer information about the sample (e.g., togenerate a diagnosis).

[0339] The database server can also be configured to communicate witheach device using commands and other signals that are interpretable bythe device. The computer-based aspects of the system can be implementedin digital electronic circuitry, or in computer hardware, firmware,software, or in combinations thereof. An apparatus of the invention,e.g., the database server, can be implemented in a computer programproduct tangibly embodied in a machine-readable storage device forexecution by a programmable processor; and method actions can beperformed by a programmable processor executing a program ofinstructions to perform functions described herein by operating on inputdata and generating output. One non-limiting example of an executionenvironment includes computers running Windows NT 4.0 (Microsoft) orbetter or Solaris 2.6 or better (Sun Microsystems) operating systems

[0340] The database server can also be interface with a network (e.g.,an intranet or the Internet). The server can receive queries from aremote system and send information (e.g., about a profile of compoundsassociated with a serum protein) to the requesting system.

[0341] A query can be used to filter the database to identify recordsthat compare favorably with a given tolerance to the query. For example,a set of mass spectroscopy peaks can be used to formulate a query. Thefilter can locate (and optionally display) records that include one ormore (e.g., all) the peaks that are present in the query.

[0342] Peptide Immobilization on NHS-Sepharose Resin

[0343] One method for producing a matrix having an immobilized peptideis as follows.

[0344] Five micromoles of each peptide were dissolved in DMSO in aminimal volume and then added to 1 ml of NHS-sepharose affinitychromatography resin (Amersham Pharmacia Biotech, Piscataway, N.J.),which had been washed once with dimethyl sulfoxide (DMSO). Theimmobilization reaction was initiated by the addition ofdiisopropylethylamine to 2% (vol/vol). After 4 hours of slow mixing on ashaker table at room temperature, the reaction was quenched by theaddition of an equal volume of 0.5 M hydroxylamine, pH 8, in water. Forthose peptides with ivDde-protected internal lysines, the hydroxylaminequench treatment also removed the ivDde-protecting group. To allow forcomplete protecting group removal, the quenched reaction was allowed toincubate overnight at room temperature. Once quenched and deprotected,the immobilized peptide-Sepharose resin was washed at least 3 times withwater to remove solvent and unbound peptide. Non-specifically boundpeptide was eluted off the resin by washing the resin at least threetimes in 30 mM phosphoric acid, pH 2. Since the NHS-Sepharose resinsurface becomes negatively charged after hydrolysis, an acidic washneutralizes the surface and removes any peptides bound non-covalently tothe surface via electrostatic interactions. After washing, the resin wasresuspended in water as a 50% v/v mixture. A 50 μl aliquot was used todetermine the ligand density on the resin by quantitative amino acidanalysis. Finally, the resin slurry was packed into 0.35 ml OMNIFIT™glass columns (3 mm×50 mm) for analytical testing.

[0345] For larger preparative columns, the amounts of peptide andSepharose were scaled up proportionally, and the final peptide Sepharosebatches were packed into larger 10 ml Omnifit columns (10 mm diameter).

EXAMPLE 1

[0346] Purification of HSA from Serum

[0347] A human serum sample was contacted to an HSA-binding peptidematrix that included the peptide ligands DX-236 and DX-321. The matrixwas washed extensively with PBS, and eluted with a basic solution, 100mM Tris, pH 9.1. Eluted material was rapidly neutralized with a buffer.An aliquot of this material was analyzed. FIG. 1 is a 2-D gel thatseparates this material.

[0348] Another aliquot of this material can be contacted to achromatography resin that includes activated maleimide. The fractionthat does not react with the resin may include compounds that dissociatefrom HSA during the elution at pH 9.1. The maleimide reacts with thefree cysteine on HSA and covalently couples the HSA to the resin. Theresin is treated with denaturants (e.g., 8M urea), and the eluant iscollected and analyzed.

[0349] In particular the eluant can be separated by 2-D electrophoresisby pI and apparent molecular weight. The 2-D gel is stained andindividual protein-staining spots are excised, digested with protease,and analyzed by MALDI-MS. The analysis of each spot is stored in adatabase.

EXAMPLE 2

[0350] Purification of HSA from Serum

[0351] HSA was purified from blood serum using a preparativeDX-236-Sepharose column (10 ml, 0.3 μmol/ml). Both the column and theserum sample were exchanged into 3 mM sodium phosphate, 20 mM NaCl, 0.1%Tween-20, pH 6.2. The 20 mM NaCl was added to the binding buffer tominimize nonspecific protein binding to the column. A 100 μl aliquot(approximately 5 mg HSA) was applied to the DX-236-Sepharose columnpreviously equilibrated in the same buffer used for dialysis. A saltgradient between 20 and 44 mM was run, and then HSA was eluted with 100mM Tris, pH 9.1. The results of the purification process are shown inTable 3. TABLE 3 Purification of HSA Using DX-236 Sepharose AffinityColumn Fraction μg HSA % Initial Initial Load 4805 100 Flowthrough 56512 Wash/Gradient 88 1.8 Elution 4003 83 Total 4656 96.8

[0352] As shown in Table 6, the column bound essentially all the HSA ina 0.1 ml serum injection (˜5 mg HSA total) and released essentially allthe bound HSA with a 100 mM Tris, pH 9.1 wash (Table 6).

EXAMPLE 3

[0353] HSA-binding Matrices

[0354] DX-232, DX-236, and DX-321 binding peptides were used foraffinity chromatography development. Each peptide was immobilized athigh density on NHS-Sepharose resin using the procedure outlined above.The peptides were immobilized via a C-terminal lysine. As determined byquantitative amino acid analysis, the ligand densities for DX-321-Sepharose, DX-236-Sepharose, and DX-232-Sepharose columns were 3.2,0.8, and 2.4 μmol/ml, respectively. Each column was tested for HSAbinding (1 mg injection) in binding buffer—3 mM sodium phosphate, 0.1%Tween-20 detergent, pH 6.2. Since some of the peptides showed a sharpincrease in K_(D) as the pH was increased to 9.1, a 100 mM Tris, pH 9.1buffer can be used to elute HSA from these columns.

[0355] For analytical affinity column testing, albumin was dissolved at1 mg/ml concentration in 3 mM sodium phosphate, pH 6.2, 0.01% Tween-20non-ionic detergent (equilibration buffer). One milliliter of albuminsolution was passed through each column (0.35 ml) previouslyequilibrated in equilibration buffer. The columns were washed with thesame equilibration buffer and then eluted with 100 mM Tris, pH 9.1 (flowrate, 0.5 ml/min for all steps). The column chromatography was carriedout using a BIO-RAD BIOLOGIC™ monitoring system (Hercules, Calif.)throughout this testing with absorbance monitoring at 280 nm.

[0356] For preparative DX-236-Sepharose affinity column (10 ml) testing,human serum was dialyzed against 3 mM phosphate, pH 6.2, 20 mM NaCl,0.01% Tween-20 non-ionic detergent (equilibration buffer). One hundredmicroliters (100 μl) of dialyzed serum were injected onto thepreparative DX-236-Sepharose chromatography column, which was previouslyequilibrated with buffer. The column was washed with the same buffer,followed by a gradient between 20 and 44 mM NaCl, and finally the HSAwas eluted with 100 mM Tris, pH 9.1. For all steps, the flow rates were5 ml/min.

[0357] Each column performed differently in the initial HSA bindingtests. Although soluble peptide DX-232 bound HSA with the highestaffinity, immobilized DX-232 on a sepharose column captured nodetectable HSA. DX-236-Sepharose, on the other hand, was the bestperformer and quantitatively bound the entire 1 mg injection (totalcapacity ≧2.7 mg/ml) (see, Table 4, below). TABLE 4 Analysis of HSAAffinity Columns Peptide in Affinity Total Column Fraction μg HSA %Initial Load Capacity DX-321 Flow through 554 55.4 Elution 370 37.0 >1.1mg/ml DX-236 Flow through 0 0 Elution 947 94.7 >2.7 mg/ml

[0358] At higher HSA loads, the same DX-236 column was capable ofbinding at least 4 mg HSA, which corresponds to a total capacity ofgreater than 11 mg/ml. DX-321-Sepharose was an intermediate performerand bound a fraction of the total material (total capacity >1.1 mg/ml).The Tris elution buffer eluted all of the bound HSA from both DX-236-and DX-321-Sepharose columns.

EXAMPLE 4

[0359] Species Specificity of Isolated HSA Binders

[0360] To test the binding specificity of DX-236 and DX-321 for HSA overother albumins, their dissociation constants (K_(D)) were determinedagainst a panel of mammalian albumins both in 3 mM sodium phosphate, pH6.2, and in PBS (10 mM sodium phosphate, 140 mM NaCl, pH 7.4). Theresults are set forth in Table 5. TABLE 5 Species Specificity Data forAffinity Columns DX-236 DX-236 DX-321 DX-321 phosphate, PBS, phosphate,PBS, pH 6.2, pH 7.4, pH 6.2, pH 7.4, % Identity O M NaCl 0.14 M NaCl O MNaCl 0.14 M NaCl Species pI to Human K_(D) (μM) K_(D) (μM) K_(D) (μM)K_(D) (μM) Human 5.67 100 1.9 11.0 0.9 84 Rhesus 5.67 93.2 1.1 23 38 82Bovine 5.60 75.6 1.1 13.3 21 >200 Goat N.D. N.D. 1.6 23 95 83 Pig 5.7575.0 0.5 12 21 >200 Rabbit 5.65 75.0 0.5 18 32 >200 Rat 5.80 73.2 1.6 2523 117 Mouse 5.53 72.0 5.5 32 >200 >200 Chicken 5.19N.D. >200 >200 >200 >200 (egg)

[0361] In the 3 mM phosphate, pH 6.2 buffer, labeled DX-236 bound to allthe albumins tested with high affinity, except for murine serum albumin(MSA). In PBS, the same affinity trend appeared with DX-236, except allthe K_(D) values were higher than for the low salt, pH 6.2 condition.

[0362] Labeled DX-321 bound each mammalian albumin with a substantiallyhigher K_(D) compared to HSA in the low salt, pH 6.2 buffer. Inparticular, MSA bound DX-321 with a K_(D) greater than 200 μM comparedto HSA, which bound DX-321 with a submicromolar K_(D). All of the othernon-human albumins also bound weakly to DX-321 and had K_(D) values atleast 10 times greater than for HSA. In PBS, however, the DX-321affinity differences between HSA and the others were less pronouncedcompared to the pH 6.2 results. As a negative control, each peptide(DX-236 and DX-321) was also tested for binding to chicken ovalbumin inboth sets of buffers and found that neither peptide showed anysignificant binding (Table 4). Chicken ovalbumin is not homologous toHSA as determined by sequence alignment analysis. This analysisindicated that immobilized DX-236 can be used to purify other mammalianalbumins.

[0363] To demonstrate this property, the same DX-236- andDX-321-Sepharose columns were tested against bovine serum albumin (BSA),goat serum albumin (GSA), and murine serum albumin (MSA) in the pH 6.2buffer. One mg of each type of albumin was injected onto each column(0.35 ml) previously equilibrated in 3 mM Phosphate, pH 6.2, 0.01%Tween-20. The columns were washed with equilibration buffer and theneluted with 100 mM Tris, pH 9.1 (flow rate, 1 ml/min). As shown in Table6 below, DX-236-Sepharose quantitatively captured all three albuminslike HSA. TABLE 6 Mammalian Serum Albumin Testing with DX-236 and DX-321DX-236 Column DX-321 Column Albumin Protein Load FT (mg) Elution (mg) FT(mg) Elution (mg) Bovine   1 mg 0 0.72 0.86 0.15 Goat 1 mg 0 0.79 0.930.11 Mouse 0.5 mg 0.05 0.59 0.49 0.13

[0364] DX-236-Sepharose can be used as a “pan-albumin” binder for theaffinity purification of nearly any mammalian albumin from serum. Theseresults indicate that DX-236 could also be used to deplete albumin fromserum samples prior to other analyses.

[0365] The data in Table 6 also show that DX-321-Sepharose captures thethree non-human albumins poorly, as is expected based on the solutionaffinity data shown in Table 4. Of the three non-human albumins, BSA wascaptured most effectively by the DX-321-Sepharose resin. About 15% ofthe BSA present in the starting material was captured and subsequentlyeluted under the same chromatography conditions that allowedquantitative capture of DX-236-Sepharose resin. Goat serum albumin (GSA)and mouse serum albumin (MSA) were even less effectively captured by theDX-321-Sepharose column than with BSA. Thus, the DX-321-Sepharose columnmay be advantageously used to purify HSA from solutions containingnon-human serum albumins.

EXAMPLE 5

[0366] Immunoglobulin Binding Peptides

[0367] Dissociation constants were determined for the followingimmunoglobulin-binding peptides, which were prepared using the Fc-regionbinding peptides of SEQ ID NOS: B57, B58, B108, B115, B124, and B143,respectively: Ac-AGSYWCKIWDVCPQSPGPEGGGK-NH₂; (SEQ ID NO:371, designatedDX392) Ac-AGKYWCNLWGVCPANPGPEGGGK-NH₂ ; (SEQ ID NO:372, designatedDX395) Ac-AGTYWCTFWELPCDPAPGPEGGGK-NH₂ ; (SEQ ID NO:373, designatedDX404) Ac-AGPHNCDDHYWYCKWFPGPEGGGK-NH₂ ; (SEQ ID NO:374, designatedDX389) Ac-AGAATCSTSYWYYQWFCTDSPGPEGGGK-NH₂ ; and (SEQ ID NO:375,designated DX398) Ac-AGYWYCWFPDRPECPLYPGPEGGGK-NH₂ . (SEQ ID NO:376,designated DX413)

[0368] Peptides were synthesized by BACHEM and then Oregon Green labeledand HPLC purified. Binding studies were performed using human plasma IgGisoforms: IgG1, IgG2, IgG3, and IgG4, obtained from Calbiochem.

[0369] Binding studies were carried out at either pH 4.0, 7.5, or 9.5,with or without salt in the following buffers:

[0370] 1) 10 mM Sodium Citrate, 0.01 % Tween 20, pH 4.0;

[0371] 2) 10 mM Sodium Citrate, 500 mM Sodium Chloride, 0.01 % Tween 20,pH 4.0;

[0372] 3) 10 mM Tris-HCl, 0.01 % Tween 20, pH 7.5;

[0373] 4) 10 mM Tris-HCl, 500 mM Sodium Chloride, 0.01 % Tween 20, pH7.5;

[0374] 5) 10 mM Sodium Bicarbonate, 0.01 % Tween 20, pH 9.5;

[0375] 6) 10 mM Sodium Bicarbonate, 500 mM Sodium Chloride, 0.01 % Tween20, pH 9.5; or

[0376] 7) TBS, 0.01 % Tween 20, pH 7.5.

[0377] Results of the binding studies are shown in Table 7. TABLE 7Summary of KD values for the IgG binding Oregon Green Labeled PeptidesK_(D)(μM) IgG pH 4.0 − pH 4.0 + pH 7.5 − pH 7.5 + pH 9.5 − pH 9.5 +Peptide isoform salt salt salt salt salt salt TBS DX389 IgG1 nb* nb nbnb nb nb nb IgG2 nb nb nb binds^(Φ) nb nb nb IgG3 2.5 ± 1.0 1.8 ± 1.4 nbbinds nb nb nb IgG4 nb nb nb nb nb nb nb DX392 IgG1 nb nb nb nb nb nb nbIgG2 nb nb nb nb nb nb nb IgG3 0.32 ± 0.08 0.6 ± 0.2 nb binds nb bindsnb IgG4 nb nb nb nb nb nb nb DX395 IgG1 nb nb nb nb nb nb nb IgG2 nb nbnb nb nb nb nb IgG3 1.0 ± 0.26 1.8 ± 1 nb 1.9 ± 0.9 nb binds nb IgG4 nbnb binds nb nb nb nb DX398 IgG1 2.4 ± 3.3 nb 4.6 ± 1.2 nb nb nb nb IgG21.8 ± 1.2 nb nb nb binds nb nb IgG3 0.02 ± 1.0  0.04 ± 0.01 nb  0.3 ±0.03 binds 0.3 ± 0.1 binds IgG4 1.6 ± 1.5 nb 3.5 ± 0.8 nb nb nb nb DX404IgG1 1.5 ± 0.8 2.0 ± 1.7 8.8 ± 4.0 nb nb nb nd IgG2   1 ± 0.4 2.0 ± 1  8.6 +3.5 nb nb nb nd IgG3 0.01 ± 0.01 0.20 ± 0.06  11 ± 5.4 3.7 ± 0.8 nbbinds nd IgG4 nb nb nb nb nb nb nd# DX413 IgG1 nb nb nb nb nb nb nd IgG2nb nb nb nb nb nb nd IgG3 0.84 ± 0.08 1.1 ± 0.2 nb nb nb nb nd IgG4 nbnb nb nb nb nb nd

[0378] The results shown in Table 7 demonstrate that DX389 specificallybinds IgG3 at pH 4.0 in either the presence or absence of salt withmoderate affinity (K_(D)≅2 μM). This interaction was not observed in thepresence or absence of salt either at pH 7.5 or 9.5.

[0379] Peptide DX392 bound IgG3 specifically at pH 4.0 both in thepresence and absence of salt and with a high affinity (K_(D)≅0.3-0.6μM). This interaction was lower at pH 7.5 and pH 9.5 in the presence ofsalt and was not observed at either pH in the absence of salt.

[0380] Peptide DX395 bound IgG3 specifically at pH 4.0 in either thepresence or absence of salt at moderate affinity (K_(D)≅1-2 μM). Theaffinity was approximately the same (K_(D)≅1.9 μM) in the presence ofsalt. This interaction was diminished at pH 9.5 in the presence of saltand was not observed at pH 7.5 or 9.5 in the absence of salt.

[0381] Peptide DX398 bound all four IgG isoforms at pH 4.0 in theabsence of salt with moderate affinity (K_(D)≅2 μM) for IgG1, IgG2, andIgG4, and high affinity (K_(D)≅0.02 μM) for IgG3. At pH 4.0 in thepresence of salt, peptide DX398 maintained a high affinity for IgG3 butdid not interact with IgG, IgG2, or IgG4.

[0382] At pH 7.5, DX398 bound IgG1 and IgG4 only in the absence of saltand in the presence of salt, only bound IgG3. At pH 9.5, this peptideonly bound IgG3 and the interaction was favored by increasing ionicstrength.

[0383] Peptide DX404 bound IgG1 and IgG2 at pH 4.0 in the presence orabsence of salt with moderate affinity (K_(D)≅2 μM) and had a higheraffinity for IgG3 (K_(D)≅0,01 μM). In the presence of salt, the affinityfor IgG3 increased to 0.2 μM. The affinity for IgG1 and IgG2 was reducedat pH 7.5 in the absence of salt and not observed in the presence ofsalt or at pH 9.5. IgG3 binding at pH 7.5 and 9.5 was favored in thepresence of salt.

[0384] Peptide DX413 bound only to IgG3 at pH 4.0 in the presence orabsence of salt with moderate affinity (K_(D)≅1.0 μM).

[0385] The data in Table 7 indicate that the peptides bind IgG withvarying isoform specificities in a pH and salt-dependent manner. Ingeneral, the peptides in Table 8 can be grouped into two “classes” basedon their specificity and mode of interaction:

[0386] Class 1 includes DX389, DX392, DX395 and DX413. Essentially thesepeptides all appear to exhibit primary specificity for IgG3. Inaddition, the interaction appears to be favored by low pH and high ionicstrength. Binding is weakest at high pH and low salt.

[0387] Class 2 includes DX398. This peptide exhibits isoform specificitythat is alterable by ionic strength. At low pH in the absence of salt,this peptide binds all IgG isoforms but in the presence of salt, it onlybinds IgG3 with very high affinity (K_(D)≅0.04-0.3 μM) at pH 4.0, 7.5,and 9.5 (See Table 7). DX404 is similar to DX398, however this peptide,unlike DX398, does not exhibit the salt-dependent IgG3 specificity at pH4.0 but does exhibit salt-dependent IgG3 specificity at pH 7.5 and 9.5.

[0388] Other embodiments are within the following claims.

1 430 1 6 PRT Artificial Sequence Examplary motif 1 Cys Xaa Xaa Xaa XaaCys 1 5 2 12 PRT Artificial Sequence Examplary motif 2 Xaa Xaa Xaa CysXaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 3 21 PRT Artificial SequenceExamplary motif 3 Ala Glu Gly Thr Gly Ser Xaa Xaa Xaa Cys Xaa Xaa XaaXaa Cys Xaa 1 5 10 15 Xaa Xaa Ala Pro Glu 20 4 12 PRT ArtificialSequence Examplary motif 4 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa XaaCys 1 5 10 5 18 PRT Artificial Sequence Examplary motif 5 Xaa Xaa XaaCys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Xaa Xaa 627 PRT Artificial Sequence Examplary motif 6 Ala Glu Gly Thr Gly Xaa XaaXaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Cys XaaXaa Xaa Xaa Pro Glu 20 25 7 6 PRT Artificial Sequence example of serumalbumin-binding agent 7 Cys Thr Ile Phe Leu Cys 1 5 8 12 PRT ArtificialSequence example of serum albumin-binding agent 8 Cys Glu Gly Lys AspMet Ile Asp Trp Val Tyr Cys 1 5 10 9 12 PRT Artificial Sequence exampleof serum albumin-binding agent 9 Cys Asp Arg Ile Ala Trp Tyr Pro Gln HisLeu Cys 1 5 10 10 10 PRT Artificial Sequence example of serumalbumin-binding agent 10 Cys Glu Pro Trp Met Leu Arg Phe Gly Cys 1 5 1011 6 PRT Artificial Sequence example of serum albumin-binding agent 11Cys Asp Gln Trp Phe Cys 1 5 12 6 PRT Artificial Sequence example ofserum albumin-binding agent 12 Cys Asn Asn Ala Leu Cys 1 5 13 6 PRTArtificial Sequence example of serum albumin-binding agent 13 Cys AspHis Phe Phe Cys 1 5 14 6 PRT Artificial Sequence example of serumalbumin-binding agent 14 Cys Trp His Phe Ser Cys 1 5 15 12 PRTArtificial Sequence example of serum albumin-binding agent 15 Cys ValThr Arg Trp Ala Asn Arg Asp Gln Gln Cys 1 5 10 16 12 PRT ArtificialSequence example of serum albumin-binding agent 16 Cys Val Thr Asp TrpAla Asn Arg His Gln His Cys 1 5 10 17 12 PRT Artificial Sequence exampleof serum albumin-binding agent 17 Cys Val Lys Asp Trp Ala Asn Arg ArgArg Gly Cys 1 5 10 18 12 PRT Artificial Sequence example of serumalbumin-binding agent 18 Cys Lys Phe Ser Trp Ile Arg Ser Pro Ala Phe Cys1 5 10 19 31 PRT Artificial Sequence serum albumin-binding agents 19 AlaGlu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 30 2027 PRT Artificial Sequence serum albumin-binding agents 20 Ala Glu GlyThr Gly Asp Arg Asn Met Cys Lys Phe Ser Trp Ile Arg 1 5 10 15 Ser ProAla Phe Cys Ala Arg Ala Asp Pro Glu 20 25 21 12 PRT Artificial Sequencetemplate sequence 21 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 510 22 16 PRT Artificial Sequence template sequence 22 Xaa Xaa Xaa CysXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 23 18 PRTArtificial Sequence template sequence 23 Xaa Xaa Xaa Cys Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15 Xaa Xaa 24 29 PRT ArtificialSequence modified serum albumin-binding agent 24 Ala Glu Gly Thr Gly AspArg Asn Met Cys Lys Phe Ser Trp Ile Arg 1 5 10 15 Ser Pro Ala Phe CysAla Arg Ala Asp Pro Glu Xaa Lys 20 25 25 14 PRT Artificial Sequencetemplate sequence 25 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa XaaXaa 1 5 10 26 585 PRT Homo sapiens 26 Asp Ala His Lys Ser Glu Val AlaHis Arg Phe Lys Asp Leu Gly Glu 1 5 10 15 Glu Asn Phe Lys Ala Leu ValLeu Ile Ala Phe Ala Gln Tyr Leu Gln 20 25 30 Gln Cys Pro Phe Glu Asp HisVal Lys Leu Val Asn Glu Val Thr Glu 35 40 45 Phe Ala Lys Thr Cys Val AlaAsp Glu Ser Ala Glu Asn Cys Asp Lys 50 55 60 Ser Leu His Thr Leu Phe GlyAsp Lys Leu Cys Thr Val Ala Thr Leu 65 70 75 80 Arg Glu Thr Tyr Gly GluMet Ala Asp Cys Cys Ala Lys Gln Glu Pro 85 90 95 Glu Arg Asn Glu Cys PheLeu Gln His Lys Asp Asp Asn Pro Asn Leu 100 105 110 Pro Arg Leu Val ArgPro Glu Val Asp Val Met Cys Thr Ala Phe His 115 120 125 Asp Asn Glu GluThr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg 130 135 140 Arg His ProTyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 TyrLys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala 165 170 175Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser 180 185190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu 195200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu ThrLys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu CysAla Asp Asp 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn GlnAsp Ser Ile Ser 260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro LeuLeu Glu Lys Ser His 275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu MetPro Ala Asp Leu Pro Ser 290 295 300 Leu Ala Ala Asp Phe Val Glu Ser LysAsp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe LeuGly Met Phe Leu Tyr Glu Tyr Ala Arg 325 330 335 Arg His Pro Asp Tyr SerVal Val Leu Leu Leu Arg Leu Ala Lys Thr 340 345 350 Tyr Glu Thr Thr LeuGlu Lys Cys Cys Ala Ala Ala Asp Pro His Glu 355 360 365 Cys Tyr Ala LysVal Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro 370 375 380 Gln Asn LeuIle Lys Gln Asn Cys Glu Leu Phe Glu Gln Leu Gly Glu 385 390 395 400 TyrLys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro 405 410 415Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys 420 425430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys 435440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr GluSer 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu ValAsp Glu Thr 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe ThrPhe His Ala Asp 500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln IleLys Lys Gln Thr Ala 515 520 525 Leu Val Glu Leu Val Lys His Lys Pro LysAla Thr Lys Glu Gln Leu 530 535 540 Lys Ala Val Met Asp Asp Phe Ala AlaPhe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr CysPhe Ala Glu Glu Gly Lys Lys Leu Val 565 570 575 Ala Ala Ser Gln Ala AlaLeu Gly Leu 580 585 27 18 PRT Artificial Sequence example of serumalbumin-binding agents 27 Ala Asp Phe Cys Glu Gly Lys Asp Met Ile AspTrp Val Tyr Cys Arg 1 5 10 15 Leu Tyr 28 18 PRT Artificial Sequenceexample of serum albumin-binding agents 28 Phe Trp Phe Cys Asp Arg IleAla Trp Tyr Pro Gln His Leu Cys Glu 1 5 10 15 Phe Leu 29 18 PRTArtificial Sequence example of serum albumin-binding agents 29 Asp TrpAsp Cys Val Thr Arg Trp Ala Asn Arg Asp Gln Gln Cys Trp 1 5 10 15 GlyPro 30 18 PRT Artificial Sequence example of serum albumin-bindingagents 30 Asp Trp Asp Cys Val Thr Arg Trp Ala Asn Arg Asp Gln Gln CysTrp 1 5 10 15 Ala Leu 31 18 PRT Artificial Sequence example of serumalbumin-binding agents 31 Asp Trp Asp Cys Val Thr Asp Trp Ala Asn ArgHis Gln His Cys Trp 1 5 10 15 Ala Leu 32 18 PRT Artificial Sequenceexample of serum albumin-binding agents 32 Asp Trp Gln Cys Val Lys AspTrp Ala Asn Arg Arg Arg Gly Cys Met 1 5 10 15 Ala Asp 33 18 PRTArtificial Sequence example of serum albumin-binding agents 33 Arg AsnMet Cys Lys Phe Ser Trp Ile Arg Ser Pro Ala Phe Cys Ala 1 5 10 15 ArgAla 34 27 PRT Artificial Sequence example of serum albumin-bindingagents 34 Ala Glu Gly Thr Gly Asp Ala Asp Phe Cys Glu Gly Lys Asp MetIle 1 5 10 15 Asp Trp Val Tyr Cys Arg Leu Tyr Asp Pro Glu 20 25 35 27PRT Artificial Sequence example of serum albumin-binding agents 35 AlaGlu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu 20 25 36 27 PRT ArtificialSequence example of serum albumin-binding agents 36 Ala Glu Gly Thr GlyAsp Asp Trp Asp Cys Val Thr Arg Trp Ala Asn 1 5 10 15 Arg Asp Gln GlnCys Trp Gly Pro Asp Pro Glu 20 25 37 27 PRT Artificial Sequence exampleof serum albumin-binding agents 37 Ala Glu Gly Thr Gly Asp Asp Trp AspCys Val Thr Arg Trp Ala Asn 1 5 10 15 Arg Asp Gln Gln Cys Trp Ala LeuAsp Pro Glu 20 25 38 27 PRT Artificial Sequence example of serumalbumin-binding agents 38 Ala Glu Gly Thr Gly Asp Asp Trp Asp Cys ValThr Asp Trp Ala Asn 1 5 10 15 Arg His Gln His Cys Trp Ala Leu Asp ProGlu 20 25 39 27 PRT Artificial Sequence example of serum albumin-bindingagents 39 Ala Glu Gly Thr Gly Asp Asp Trp Gln Cys Val Lys Asp Trp AlaAsn 1 5 10 15 Arg Arg Arg Gly Cys Met Ala Asp Asp Pro Glu 20 25 40 27PRT Artificial Sequence example of serum albumin-binding agents 40 AlaGlu Gly Thr Gly Asp Arg Asn Met Cys Lys Phe Ser Trp Ile Arg 1 5 10 15Ser Pro Ala Phe Cys Ala Arg Ala Asp Pro Glu 20 25 41 12 PRT ArtificialSequence example of serum albumin-binding agents 41 Cys Asp Arg Ile AlaTrp Tyr Pro Gln His Ala Cys 1 5 10 42 12 PRT Artificial Sequence exampleof serum albumin-binding agents 42 Cys Asp Arg Ile Ala Trp Tyr Pro GlnAla Leu Cys 1 5 10 43 12 PRT Artificial Sequence example of serumalbumin-binding agents 43 Cys Asp Arg Ile Ala Trp Tyr Pro Ala His LeuCys 1 5 10 44 12 PRT Artificial Sequence example of serumalbumin-binding agents 44 Cys Asp Arg Ile Ala Trp Tyr Ala Gln His LeuCys 1 5 10 45 12 PRT Artificial Sequence example of serumalbumin-binding agents 45 Cys Asp Arg Ile Ala Trp Ala Pro Gln His LeuCys 1 5 10 46 12 PRT Artificial Sequence example of serumalbumin-binding agents 46 Cys Asp Arg Ile Ala Ala Tyr Pro Gln His LeuCys 1 5 10 47 12 PRT Artificial Sequence example of serumalbumin-binding agents 47 Cys Asp Arg Ala Ala Trp Tyr Pro Gln His LeuCys 1 5 10 48 12 PRT Artificial Sequence example of serumalbumin-binding agents 48 Cys Asp Ala Ile Ala Trp Tyr Pro Gln His LeuCys 1 5 10 49 12 PRT Artificial Sequence example of serumalbumin-binding agents 49 Cys Ala Arg Ile Ala Trp Tyr Pro Gln His LeuCys 1 5 10 50 18 PRT Artificial Sequence example of serumalbumin-binding agents 50 Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr ProGln His Leu Cys Glu 1 5 10 15 Phe Ala 51 18 PRT Artificial Sequenceexample of serum albumin-binding agents 51 Phe Trp Phe Cys Asp Arg IleAla Trp Tyr Pro Gln His Leu Cys Glu 1 5 10 15 Ala Leu 52 18 PRTArtificial Sequence example of serum albumin-binding agents 52 Phe TrpPhe Cys Asp Arg Ile Ala Trp Tyr Pro Gln His Leu Cys Ala 1 5 10 15 PheLeu 53 18 PRT Artificial Sequence example of serum albumin-bindingagents 53 Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr Pro Gln His Ala CysGlu 1 5 10 15 Phe Leu 54 18 PRT Artificial Sequence example of serumalbumin-binding agents 54 Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr ProGln Ala Leu Cys Glu 1 5 10 15 Phe Leu 55 18 PRT Artificial Sequenceexample of serum albumin-binding agents 55 Phe Trp Phe Cys Asp Arg IleAla Trp Tyr Pro Ala His Leu Cys Glu 1 5 10 15 Phe Leu 56 18 PRTArtificial Sequence example of serum albumin-binding agents 56 Phe TrpPhe Cys Asp Arg Ile Ala Trp Tyr Ala Gln His Leu Cys Glu 1 5 10 15 PheLeu 57 18 PRT Artificial Sequence example of serum albumin-bindingagents 57 Phe Trp Phe Cys Asp Arg Ile Ala Trp Ala Pro Gln His Leu CysGlu 1 5 10 15 Phe Leu 58 18 PRT Artificial Sequence example of serumalbumin-binding agents 58 Phe Trp Phe Cys Asp Arg Ile Ala Ala Tyr ProGln His Leu Cys Glu 1 5 10 15 Phe Leu 59 18 PRT Artificial Sequenceexample of serum albumin-binding agents 59 Phe Trp Phe Cys Asp Arg AlaAla Trp Tyr Pro Gln His Leu Cys Glu 1 5 10 15 Phe Leu 60 18 PRTArtificial Sequence example of serum albumin-binding agents 60 Phe TrpPhe Cys Asp Ala Ile Ala Trp Tyr Pro Gln His Leu Cys Glu 1 5 10 15 PheLeu 61 18 PRT Artificial Sequence example of serum albumin-bindingagents 61 Phe Trp Phe Cys Ala Arg Ile Ala Trp Tyr Pro Gln His Leu CysGlu 1 5 10 15 Phe Leu 62 18 PRT Artificial Sequence example of serumalbumin-binding agents 62 Phe Trp Ala Cys Asp Arg Ile Ala Trp Tyr ProGln His Leu Cys Glu 1 5 10 15 Phe Leu 63 18 PRT Artificial Sequenceexample of serum albumin-binding agents 63 Phe Ala Phe Cys Asp Arg IleAla Trp Tyr Pro Gln His Leu Cys Glu 1 5 10 15 Phe Leu 64 18 PRTArtificial Sequence example of serum albumin-binding agents 64 Ala TrpPhe Cys Asp Arg Ile Ala Trp Tyr Pro Gln His Leu Cys Glu 1 5 10 15 PheLeu 65 27 PRT Artificial Sequence example of serum albumin-bindingagents 65 Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala TrpTyr 1 5 10 15 Pro Gln His Leu Cys Glu Phe Leu Ala Pro Glu 20 25 66 27PRT Artificial Sequence example of serum albumin-binding agents 66 AlaGlu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15Pro Gln His Leu Cys Glu Phe Ala Asp Pro Glu 20 25 67 27 PRT ArtificialSequence example of serum albumin-binding agents 67 Ala Glu Gly Thr GlyAsp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15 Pro Gln His LeuCys Glu Ala Leu Asp Pro Glu 20 25 68 27 PRT Artificial Sequence exampleof serum albumin-binding agents 68 Ala Glu Gly Thr Gly Asp Phe Trp PheCys Asp Arg Ile Ala Trp Tyr 1 5 10 15 Pro Gln His Leu Cys Ala Phe LeuAsp Pro Glu 20 25 69 27 PRT Artificial Sequence example of serumalbumin-binding agents 69 Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys AspArg Ile Ala Trp Tyr 1 5 10 15 Pro Gln His Ala Cys Glu Phe Leu Asp ProGlu 20 25 70 27 PRT Artificial Sequence example of serum albumin-bindingagents 70 Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala TrpTyr 1 5 10 15 Pro Gln Ala Leu Cys Glu Phe Leu Asp Pro Glu 20 25 71 27PRT Artificial Sequence example of serum albumin-binding agents 71 AlaGlu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15Pro Ala His Leu Cys Glu Phe Leu Asp Pro Glu 20 25 72 27 PRT ArtificialSequence example of serum albumin-binding agents 72 Ala Glu Gly Thr GlyAsp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15 Ala Gln His LeuCys Glu Phe Leu Asp Pro Glu 20 25 73 27 PRT Artificial Sequence exampleof serum albumin-binding agents 73 Ala Glu Gly Thr Gly Asp Phe Trp PheCys Asp Arg Ile Ala Trp Ala 1 5 10 15 Pro Gln His Leu Cys Glu Phe LeuAsp Pro Glu 20 25 74 27 PRT Artificial Sequence example of serumalbumin-binding agents 74 Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys AspArg Ile Ala Ala Tyr 1 5 10 15 Pro Gln His Leu Cys Glu Phe Leu Asp ProGlu 20 25 75 27 PRT Artificial Sequence example of serum albumin-bindingagents 75 Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ala Ala TrpTyr 1 5 10 15 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu 20 25 76 27PRT Artificial Sequence example of serum albumin-binding agents 76 AlaGlu Gly Thr Gly Asp Phe Trp Phe Cys Asp Ala Ile Ala Trp Tyr 1 5 10 15Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu 20 25 77 27 PRT ArtificialSequence example of serum albumin-binding agents 77 Ala Glu Gly Thr GlyAsp Phe Trp Phe Cys Ala Arg Ile Ala Trp Tyr 1 5 10 15 Pro Gln His LeuCys Glu Phe Leu Asp Pro Glu 20 25 78 27 PRT Artificial Sequence exampleof serum albumin-binding agents 78 Ala Glu Gly Thr Gly Asp Phe Trp AlaCys Asp Arg Ile Ala Trp Tyr 1 5 10 15 Pro Gln His Leu Cys Glu Phe LeuAsp Pro Glu 20 25 79 27 PRT Artificial Sequence example of serumalbumin-binding agents 79 Ala Glu Gly Thr Gly Asp Phe Ala Phe Cys AspArg Ile Ala Trp Tyr 1 5 10 15 Pro Gln His Leu Cys Glu Phe Leu Asp ProGlu 20 25 80 27 PRT Artificial Sequence example of serum albumin-bindingagents 80 Ala Glu Gly Thr Gly Asp Ala Trp Phe Cys Asp Arg Ile Ala TrpTyr 1 5 10 15 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu 20 25 81 27PRT Artificial Sequence example of serum albumin-binding agents 81 AlaGlu Gly Thr Gly Ala Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu 20 25 82 31 PRT ArtificialSequence example of serum albumin-binding agents 82 Ala Glu Gly Thr GlyAsp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15 Pro Gln His LeuCys Glu Phe Leu Ala Pro Glu Gly Gly Gly Lys 20 25 30 83 31 PRTArtificial Sequence example of serum albumin-binding agents 83 Ala GluGly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15 ProGln His Leu Cys Glu Phe Ala Asp Pro Glu Gly Gly Gly Lys 20 25 30 84 31PRT Artificial Sequence example of serum albumin-binding agents 84 AlaGlu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 10 15Pro Gln His Leu Cys Glu Ala Leu Asp Pro Glu Gly Gly Gly Lys 20 25 30 8531 PRT Artificial Sequence example of serum albumin-binding agents 85Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Ala Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3086 31 PRT Artificial Sequence example of serum albumin-binding agents 86Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln His Ala Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3087 31 PRT Artificial Sequence example of serum albumin-binding agents 87Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln Ala Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3088 31 PRT Artificial Sequence example of serum albumin-binding agents 88Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Ala His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3089 31 PRT Artificial Sequence example of serum albumin-binding agents 89Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Ala Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3090 31 PRT Artificial Sequence example of serum albumin-binding agents 90Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Trp Ala 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3091 31 PRT Artificial Sequence example of serum albumin-binding agents 91Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ile Ala Ala Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3092 31 PRT Artificial Sequence example of serum albumin-binding agents 92Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Arg Ala Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3093 31 PRT Artificial Sequence example of serum albumin-binding agents 93Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Asp Ala Ile Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3094 31 PRT Artificial Sequence example of serum albumin-binding agents 94Ala Glu Gly Thr Gly Asp Phe Trp Phe Cys Ala Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3095 31 PRT Artificial Sequence example of serum albumin-binding agents 95Ala Glu Gly Thr Gly Asp Phe Trp Ala Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3096 31 PRT Artificial Sequence example of serum albumin-binding agents 96Ala Glu Gly Thr Gly Asp Phe Ala Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3097 31 PRT Artificial Sequence example of serum albumin-binding agents 97Ala Glu Gly Thr Gly Asp Ala Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3098 31 PRT Artificial Sequence example of serum albumin-binding agents 98Ala Glu Gly Thr Gly Ala Phe Trp Phe Cys Asp Arg Ile Ala Trp Tyr 1 5 1015 Pro Gln His Leu Cys Glu Phe Leu Asp Pro Glu Gly Gly Gly Lys 20 25 3099 14 PRT Artificial Sequence example of serum albumin-binding agents 99Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 100 18PRT Artificial Sequence example of serum albumin-binding agents 100 AlaGly Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15Gly Thr 101 10 PRT Artificial Sequence example of serum albumin-bindingagents 101 Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys 1 5 10 102 16 PRTArtificial Sequence example of serum albumin-binding agents 102 Xaa XaaXaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa Xaa Xaa 1 5 10 15 103 20PRT Artificial Sequence example of serum albumin-binding agents 103 GlySer Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 10 15Xaa Xaa Ala Pro 20 104 20 PRT Artificial Sequence example of serumalbumin-binding agents 104 Pro Thr Val Val Gln Pro Lys Phe His Ala PheThr His Glu Asp Leu 1 5 10 15 Leu Trp Ile Phe 20 105 20 PRT ArtificialSequence example of serum albumin-binding agents 105 Leu Lys Ser Gln MetVal His Ala Leu Pro Ala Ala Ser Leu His Asp 1 5 10 15 Gln His Glu Leu 20106 20 PRT Artificial Sequence example of serum albumin-binding agents106 Ser Gln Val Gln Gly Thr Pro Asp Leu Gln Phe Thr Val Arg Asp Phe 1 510 15 Ile Tyr Met Phe 20 107 12 PRT Artificial Sequence example of serumalbumin-binding agents 107 Cys Gln Thr Thr Trp Pro Phe Thr Met Met GlnCys 1 5 10 108 12 PRT Artificial Sequence example of serumalbumin-binding agents 108 Cys Val Thr Met Trp Pro Phe Glu Gln Ile PheCys 1 5 10 109 12 PRT Artificial Sequence example of serumalbumin-binding agents 109 Cys Phe Thr Tyr Tyr Pro Phe Thr Thr Phe SerCys 1 5 10 110 12 PRT Artificial Sequence example of serumalbumin-binding agents 110 Cys Trp Thr Lys Phe Pro Phe Asp Leu Val TrpCys 1 5 10 111 12 PRT Artificial Sequence example of serumalbumin-binding agents 111 Cys Val Ser Tyr Trp Pro His Phe Val Pro ValCys 1 5 10 112 12 PRT Artificial Sequence example of serumalbumin-binding agents 112 Cys Tyr Ile Ser Phe Pro Phe Asp Gln Met TyrCys 1 5 10 113 12 PRT Artificial Sequence example of serumalbumin-binding agents 113 Cys Ser Val Gln Tyr Pro Phe Glu Val Val ValCys 1 5 10 114 12 PRT Artificial Sequence example of serumalbumin-binding agents 114 Cys Trp Thr Gln Tyr Pro Phe Asp His Ser ThrCys 1 5 10 115 12 PRT Artificial Sequence example of serumalbumin-binding agents 115 Cys Ile Thr Trp Pro Phe Lys Arg Pro Trp ProCys 1 5 10 116 12 PRT Artificial Sequence example of serumalbumin-binding agents 116 Cys Ile Ser Trp Pro Phe Glu Met Pro Phe HisCys 1 5 10 117 12 PRT Artificial Sequence example of serumalbumin-binding agents 117 Cys Ile Thr Trp Pro Phe Lys Arg Pro Trp ProCys 1 5 10 118 12 PRT Artificial Sequence example of serumalbumin-binding agents 118 Cys Ile Thr Tyr Pro Phe His Glu Met Phe ProCys 1 5 10 119 12 PRT Artificial Sequence example of serumalbumin-binding agents 119 Cys Ile Thr Trp Pro Phe Gln Thr Ser Tyr ProCys 1 5 10 120 12 PRT Artificial Sequence example of serumalbumin-binding agents 120 Cys Lys Phe Ser Trp Ile Arg Ser Pro Ala PheCys 1 5 10 121 12 PRT Artificial Sequence example of serumalbumin-binding agents 121 Cys Trp Ile Val Asp Glu Asp Gly Thr Lys TrpCys 1 5 10 122 12 PRT Artificial Sequence example of serumalbumin-binding agents 122 Cys Asp Ser Ala Tyr Trp Gln Glu Ile Pro AlaCys 1 5 10 123 8 PRT Artificial Sequence example of serumalbumin-binding agents 123 Cys Leu Trp Asp Pro Met Leu Cys 1 5 124 12PRT Artificial Sequence example of serum albumin-binding agents 124 CysGlu His Pro Tyr Trp Thr Glu Val Asp Lys Cys 1 5 10 125 12 PRT ArtificialSequence example of serum albumin-binding agents 125 Cys Asp Thr Pro TyrTrp Arg Asp Leu Trp Gln Cys 1 5 10 126 12 PRT Artificial Sequenceexample of serum albumin-binding agents 126 Cys Gln Leu Pro Tyr Met SerThr Pro Glu Phe Cys 1 5 10 127 12 PRT Artificial Sequence example ofserum albumin-binding agents 127 Cys Gly Arg Gly Phe Asp Lys Glu Ser IleTyr Cys 1 5 10 128 12 PRT Artificial Sequence example of serumalbumin-binding agents 128 Cys Val Thr Tyr Ile Gly Thr Trp Glu Thr ValCys 1 5 10 129 12 PRT Artificial Sequence example of serumalbumin-binding agents 129 Cys Thr Asp Thr Asn Trp Ser Trp Met Phe AspCys 1 5 10 130 12 PRT Artificial Sequence example of serumalbumin-binding agents 130 Cys Thr Leu Glu Ile Gly Thr Trp Phe Val PheCys 1 5 10 131 12 PRT Artificial Sequence example of serumalbumin-binding agents 131 Cys Lys Ile Ala Leu Phe Gln His Phe Glu ValCys 1 5 10 132 12 PRT Artificial Sequence example of serumalbumin-binding agents 132 Cys Ile Lys Leu Tyr Gly Leu Gly His Met TyrCys 1 5 10 133 12 PRT Artificial Sequence example of serumalbumin-binding agents 133 Cys Glu Met Gln Ser Ile Ile Pro Trp Trp GluCys 1 5 10 134 12 PRT Artificial Sequence example of serumalbumin-binding agents 134 Cys Val Glu Lys Tyr Tyr Trp Asp Val Leu IleCys 1 5 10 135 11 PRT Artificial Sequence example of serumalbumin-binding agents 135 Cys Pro His Gly Arg Tyr Ser Met Phe Pro Cys 15 10 136 12 PRT Artificial Sequence example of serum albumin-bindingagents 136 Cys Asn Val Arg Trp Thr Asp Thr Pro Tyr Trp Cys 1 5 10 137 12PRT Artificial Sequence example of serum albumin-binding agents 137 CysThr Tyr Asp Pro Ile Ala Asp Leu Leu Phe Cys 1 5 10 138 10 PRT ArtificialSequence example of serum albumin-binding agents 138 Cys Met Asp Trp ProAsn His Arg Asp Cys 1 5 10 139 10 PRT Artificial Sequence example ofserum albumin-binding agents 139 Cys Phe Pro Ile His Leu Thr Met Phe Cys1 5 10 140 10 PRT Artificial Sequence example of serum albumin-bindingagents 140 Cys Gln Thr Ser Phe Thr Asn Tyr Trp Cys 1 5 10 141 9 PRTArtificial Sequence example of serum albumin-binding agents 141 Cys MetGlu Phe Gly Pro Asp Asp Cys 1 5 142 8 PRT Artificial Sequence example ofserum albumin-binding agents 142 Cys Ser Trp Asp Pro Ile Phe Cys 1 5 1438 PRT Artificial Sequence example of serum albumin-binding agents 143Cys Ala Trp Asp Pro Leu Val Cys 1 5 144 8 PRT Artificial Sequenceexample of serum albumin-binding agents 144 Cys His Ile Tyr Asp Trp PheCys 1 5 145 8 PRT Artificial Sequence example of serum albumin-bindingagents 145 Cys Leu Trp Asp Pro Met Ile Cys 1 5 146 8 PRT ArtificialSequence example of serum albumin-binding agents 146 Cys Ser Pro Pro GlyLys Thr Cys 1 5 147 8 PRT Artificial Sequence example of serumalbumin-binding agents 147 Cys Thr Phe Trp Gln Tyr Trp Cys 1 5 148 8 PRTArtificial Sequence example of serum albumin-binding agents 148 Cys MetPhe Glu Leu Pro Phe Cys 1 5 149 8 PRT Artificial Sequence example ofserum albumin-binding agents 149 Cys Phe Ser Lys Pro Asp Gln Cys 1 5 1508 PRT Artificial Sequence example of serum albumin-binding agents 150Cys Phe Tyr Gln Trp Trp Gly Cys 1 5 151 8 PRT Artificial Sequenceexample of serum albumin-binding agents 151 Cys Thr Trp Asp Pro Ile PheCys 1 5 152 6 PRT Artificial Sequence example of serum albumin-bindingagents 152 Cys Trp Leu Tyr Asp Cys 1 5 153 6 PRT Artificial Sequenceexample of serum albumin-binding agents 153 Cys Asp Lys Tyr Gly Cys 1 5154 6 PRT Artificial Sequence example of serum albumin-binding agents154 Cys Ser Lys Asp Thr Cys 1 5 155 17 PRT Artificial Sequence exampleof serum albumin-binding agents 155 Leu Arg Asp Cys Gln Thr Thr Trp ProPhe Met Met Gln Cys Pro Asn 1 5 10 15 Asn 156 18 PRT Artificial Sequenceexample of serum albumin-binding agents 156 Asn Arg Glu Cys Val Thr MetTrp Pro Phe Glu Gln Ile Phe Cys Pro 1 5 10 15 Trp Pro 157 18 PRTArtificial Sequence example of serum albumin-binding agents 157 Leu ArgSer Cys Phe Thr Tyr Tyr Pro Phe Thr Thr Phe Ser Cys Ser 1 5 10 15 ProAla 158 18 PRT Artificial Sequence example of serum albumin-bindingagents 158 Leu Ser His Cys Trp Thr Lys Phe Pro Phe Asp Leu Val Trp CysAsp 1 5 10 15 Ser Pro 159 18 PRT Artificial Sequence example of serumalbumin-binding agents 159 Leu Arg Met Cys Val Ser Tyr Trp Pro His PheVal Pro Val Cys Glu 1 5 10 15 Asn Pro 160 18 PRT Artificial Sequenceexample of serum albumin-binding agents 160 Leu Arg Asp Cys Tyr Ile SerPhe Pro Phe Asp Gln Met Tyr Cys Ser 1 5 10 15 His Phe 161 18 PRTArtificial Sequence example of serum albumin-binding agents 161 Phe ArgHis Cys Ser Val Gln Tyr Pro Phe Glu Val Val Val Cys Pro 1 5 10 15 AlaAsn 162 18 PRT Artificial Sequence example of serum albumin-bindingagents 162 Leu Arg Asn Cys Trp Thr Gln Tyr Pro Phe Asp His Ser Thr CysSer 1 5 10 15 Pro Asn 163 17 PRT Artificial Sequence example of serumalbumin-binding agents 163 Asp Ser Met Cys Ile Thr Trp Pro Phe Lys ArgPro Trp Pro Cys Ala 1 5 10 15 Asn 164 18 PRT Artificial Sequence exampleof serum albumin-binding agents 164 Ala Phe Met Cys Ile Ser Trp Pro PheGlu Met Pro Phe His Cys Ser 1 5 10 15 Pro Asp 165 18 PRT ArtificialSequence example of serum albumin-binding agents 165 Asp Ser Met Cys IleThr Trp Pro Phe Lys Arg Pro Trp Pro Cys Ala 1 5 10 15 Asn Pro 166 18 PRTArtificial Sequence example of serum albumin-binding agents 166 Trp AspLeu Cys Ile Thr Tyr Pro Phe His Glu Met Phe Pro Cys Glu 1 5 10 15 AspGly 167 18 PRT Artificial Sequence example of serum albumin-bindingagents 167 Gly Gly Glu Cys Ile Thr Trp Pro Phe Gln Thr Ser Tyr Pro CysThr 1 5 10 15 Asn Gly 168 18 PRT Artificial Sequence example of serumalbumin-binding agents 168 Arg Asn Met Cys Lys Phe Ser Trp Ile Arg SerPro Ala Phe Cys Ala 1 5 10 15 Arg Ala 169 17 PRT Artificial Sequenceexample of serum albumin-binding agents 169 Phe Ser Leu Cys Trp Ile ValAsp Glu Asp Gly Thr Lys Trp Cys Leu 1 5 10 15 Pro 170 18 PRT ArtificialSequence example of serum albumin-binding agents 170 Arg Trp Phe Cys AspSer Ala Tyr Trp Gln Glu Ile Pro Ala Cys Ala 1 5 10 15 Arg Asp 171 14 PRTArtificial Sequence example of serum albumin-binding agents 171 Arg TrpTyr Cys Leu Trp Asp Pro Met Leu Cys Met Ser Asp 1 5 10 172 18 PRTArtificial Sequence example of serum albumin-binding agents 172 Ala TrpTyr Cys Glu His Pro Tyr Trp Thr Glu Val Asp Lys Cys His 1 5 10 15 SerSer 173 18 PRT Artificial Sequence example of serum albumin-bindingagents 173 Ser Asp Phe Cys Asp Thr Pro Tyr Trp Arg Asp Leu Trp Gln CysAsn 1 5 10 15 Ser Pro 174 18 PRT Artificial Sequence example of serumalbumin-binding agents 174 Leu Pro Trp Cys Gln Leu Pro Tyr Met Ser ThrPro Glu Phe Cys Ile 1 5 10 15 Arg Pro 175 18 PRT Artificial Sequenceexample of serum albumin-binding agents 175 Tyr His Val Cys Gly Arg GlyPhe Asp Lys Glu Ser Ile Tyr Cys Lys 1 5 10 15 Phe Leu 176 17 PRTArtificial Sequence example of serum albumin-binding agents 176 Ser PheCys Val Thr Tyr Ile Gly Thr Trp Glu Thr Val Cys Lys Arg 1 5 10 15 Ser177 18 PRT Artificial Sequence example of serum albumin-binding agent177 Asn Asp Gly Cys Thr Asp Thr Asn Trp Ser Trp Met Phe Asp Cys Pro 1 510 15 Pro Leu 178 18 PRT Artificial Sequence example of serumalbumin-binding agent 178 Trp Arg Asp Cys Thr Leu Glu Ile Gly Thr TrpPhe Val Phe Cys Lys 1 5 10 15 Gly Ser 179 18 PRT Artificial Sequenceexample of serum albumin-binding agent 179 Ser Pro Tyr Cys Lys Ile AlaLeu Phe Gln His Phe Glu Val Cys Ala 1 5 10 15 Ala Asp 180 18 PRTArtificial Sequence example of serum albumin-binding agent 180 Arg HisTrp Cys Ile Lys Leu Tyr Gly Leu Gly His Met Tyr Cys Asn 1 5 10 15 ArgSer 181 18 PRT Artificial Sequence serum albumin-binding agent 181 AspHis Ala Cys Glu Met Gln Ser Ile Ile Pro Trp Trp Glu Cys Tyr 1 5 10 15Pro His 182 18 PRT Artificial Sequence example of serum albumin-bindingagent 182 Pro Arg Ser Cys Val Glu Lys Tyr Tyr Trp Asp Val Leu Ile CysGly 1 5 10 15 Phe Phe 183 17 PRT Artificial Sequence example of serumalbumin-binding agent 183 Phe His Thr Cys Pro His Gly Arg Tyr Ser MetPhe Pro Cys Asp Tyr 1 5 10 15 Trp 184 18 PRT Artificial Sequence exampleof serum albumin-binding agent 184 His Gly Trp Cys Asn Val Arg Trp ThrAsp Thr Pro Tyr Trp Cys Ala 1 5 10 15 Phe Ser 185 18 PRT ArtificialSequence example of serum albumin-binding agent 185 Tyr Arg Val Cys ThrTyr Asp Pro Ile Ala Asp Leu Leu Phe Cys Pro 1 5 10 15 Phe Asn 186 16 PRTArtificial Sequence example of serum albumin-binding agent 186 Arg SerPhe Cys Met Asp Trp Pro Asn His Arg Asp Cys Asp Tyr Ser 1 5 10 15 187 16PRT Artificial Sequence example of serum albumin-binding agent 187 PheTrp Asp Cys Phe Pro Ile His Leu Thr Met Phe Cys Asp Arg Phe 1 5 10 15188 16 PRT Artificial Sequence example of serum albumin-binding agent188 Tyr Leu Tyr Cys Gln Thr Ser Phe Thr Asn Tyr Trp Cys Ala Phe His 1 510 15 189 15 PRT Artificial Sequence example of serum albumin-bindingagent 189 Gly Leu Tyr Cys Met Glu Phe Gly Pro Asp Asp Cys Ala Trp His 15 10 15 190 14 PRT Artificial Sequence example of serum albumin-bindingagent 190 Lys Asn Phe Cys Ser Trp Asp Pro Ile Phe Cys Gly Ile His 1 5 10191 14 PRT Artificial Sequence example of serum albumin-binding agent191 Lys Trp Tyr Cys Ala Trp Asp Pro Leu Val Cys Glu Ile Phe 1 5 10 19214 PRT Artificial Sequence example of serum albumin-binding agent 192Trp Thr Thr Cys His Ile Tyr Asp Trp Phe Cys Ser Ser Ser 1 5 10 193 14PRT Artificial Sequence example of serum albumin-binding agent 193 GlnTrp Tyr Cys Leu Trp Asp Pro Met Ile Cys Gly Leu Ile 1 5 10 194 14 PRTArtificial Sequence example of serum albumin-binding agent 194 Gln ThrAsn Cys Ser Pro Pro Gly Lys Thr Cys Asp Lys Asn 1 5 10 195 13 PRTArtificial Sequence example of serum albumin-binding agent 195 Ala IleCys Thr Phe Trp Gln Tyr Trp Cys Leu Glu Pro 1 5 10 196 14 PRT ArtificialSequence example of serum albumin-binding agent 196 Phe Glu Trp Cys MetPhe Glu Leu Pro Phe Cys Ser Trp Pro 1 5 10 197 14 PRT ArtificialSequence example of serum albumin-binding agent 197 Gln Glu Gly Cys PheSer Lys Pro Asp Gln Cys Lys Val Met 1 5 10 198 14 PRT ArtificialSequence example of serum albumin-binding agent 198 Leu Glu Tyr Cys PheTyr Gln Trp Trp Gly Cys Pro His Ala 1 5 10 199 14 PRT ArtificialSequence example of serum albumin-binding agent 199 Tyr Gln Phe Cys ThrTrp Asp Pro Ile Phe Cys Gly Trp His 1 5 10 200 12 PRT ArtificialSequence example of serum albumin-binding agent 200 Leu Trp Asp Cys TrpLeu Tyr Asp Cys Glu Gly Asn 1 5 10 201 12 PRT Artificial Sequenceexample of serum albumin-binding agent 201 Val His Ser Cys Asp Lys TyrGly Cys Val Asn Ala 1 5 10 202 12 PRT Artificial Sequence example ofserum albumin-binding agent 202 Phe Glu His Cys Ser Lys Asp Thr Cys SerGly Asn 1 5 10 203 12 PRT Artificial Sequence example of serumalbumin-binding agent 203 Val Ala Trp Cys Thr Ile Phe Leu Cys Leu AspVal 1 5 10 204 12 PRT Artificial Sequence example of serumalbumin-binding agent 204 Phe Lys Ile Cys Asp Gln Trp Phe Cys Leu MetPro 1 5 10 205 12 PRT Artificial Sequence example of serumalbumin-binding agent 205 His Val Gly Cys Asn Asn Ala Leu Cys Met GlnTyr 1 5 10 206 12 PRT Artificial Sequence example of serumalbumin-binding agent 206 Trp Lys Val Cys Asp His Phe Phe Cys Leu SerPro 1 5 10 207 12 PRT Artificial Sequence example of serumalbumin-binding agent 207 Asn His Gly Cys Trp His Phe Ser Cys Ile TrpAsp 1 5 10 208 16 PRT Artificial Sequence example of serumalbumin-binding agent 208 Phe Arg Asn Cys Glu Pro Trp Met Leu Arg PheGly Cys Asn Pro Arg 1 5 10 15 209 18 PRT Artificial Sequence example ofserum albumin-binding agent 209 Ala Asp Phe Cys Glu Gly Lys Asp Met IleAsp Trp Val Tyr Cys Arg 1 5 10 15 Leu Tyr 210 19 PRT Artificial Sequenceexample of serum albumin-binding agent 210 Phe Trp Phe Cys Asp Arg IleAla Trp Tyr Pro Gln His Leu Cys Glu 1 5 10 15 Phe Leu Asp 211 18 PRTArtificial Sequence example of serum albumin-binding agent 211 Asp TrpAsp Cys Val Thr Arg Trp Ala Asn Arg Asp Gln Gln Cys Trp 1 5 10 15 GlyPro 212 18 PRT Artificial Sequence example of serum albumin-bindingagent 212 Asp Trp Asp Cys Val Thr Arg Trp Ala Asn Arg Asp Gln Gln CysTrp 1 5 10 15 Ala Leu 213 18 PRT Artificial Sequence example of serumalbumin-binding agent 213 Asp Trp Asp Cys Val Thr Asp Trp Ala Asn ArgHis Gln His Cys Trp 1 5 10 15 Ala Leu 214 18 PRT Artificial Sequenceexample of serum albumin-binding agent 214 Asp Trp Gln Cys Val Lys AspTrp Ala Asn Arg Arg Arg Gly Cys Met 1 5 10 15 Ala Asp 215 20 PRTArtificial Sequence example of serum albumin-binding agent 215 Arg AsnMet Cys Lys Phe Ser Trp Ile Arg Ser Pro Ala Phe Cys Ala 1 5 10 15 ArgAla Asp Pro 20 216 26 PRT Artificial Sequence serum albumin-bindingagent 216 Gly Asp Leu Arg Asp Cys Gln Thr Thr Trp Pro Phe Thr Met MetGln 1 5 10 15 Cys Pro Asn Asn Asp Pro Gly Gly Gly Lys 20 25 217 26 PRTArtificial Sequence serum albumin-binding agent 217 Gly Asp Asn Arg GluCys Val Thr Met Trp Pro Phe Glu Gln Ile Phe 1 5 10 15 Cys Pro Trp ProAsp Pro Gly Gly Gly Lys 20 25 218 26 PRT Artificial Sequence serumalbumin-binding agent 218 Gly Asp Leu Arg Ser Cys Phe Thr Tyr Tyr ProPhe Thr Thr Phe Ser 1 5 10 15 Cys Ser Pro Ala Asp Pro Gly Gly Gly Lys 2025 219 25 PRT Artificial Sequence serum albumin-binding agent 219 GlyAsp Asp Ser Met Cys Ile Thr Trp Pro Phe Lys Arg Pro Trp Pro 1 5 10 15Cys Ala Asn Asp Pro Gly Gly Gly Lys 20 25 220 26 PRT Artificial Sequenceserum albumin-binding agent 220 Gly Asp Arg Asn Met Cys Lys Phe Ser TrpIle Arg Ser Pro Ala Phe 1 5 10 15 Cys Ala Arg Ala Asp Pro Gly Gly GlyLys 20 25 221 25 PRT Artificial Sequence serum albumin-binding agent 221Gly Asp Phe Ser Leu Cys Trp Ile Val Asp Glu Asp Gly Thr Lys Trp 1 5 1015 Cys Leu Pro Asp Pro Gly Gly Gly Lys 20 25 222 26 PRT ArtificialSequence serum albumin-binding agent 222 Gly Asp Arg Trp Phe Cys Asp SerAla Tyr Trp Gln Glu Ile Pro Ala 1 5 10 15 Cys Ala Arg Asp Asp Pro GlyGly Gly Lys 20 25 223 26 PRT Artificial Sequence serum albumin-bindingagent 223 Gly Asp Ser Asp Phe Cys Asp Thr Pro Tyr Trp Arg Asp Leu TrpGln 1 5 10 15 Cys Asn Ser Pro Asp Pro Gly Gly Gly Lys 20 25 224 25 PRTArtificial Sequence serum albumin-binding agent 224 Gly Asp Ser Phe CysVal Thr Tyr Ile Gly Thr Trp Glu Thr Val Cys 1 5 10 15 Lys Arg Ser AspPro Gly Gly Gly Lys 20 25 225 26 PRT Artificial Sequence serumalbumin-binding agent 225 Gly Asp Asn Asp Gly Cys Thr Asp Thr Asn TrpSer Trp Met Phe Asp 1 5 10 15 Cys Pro Pro Leu Asp Pro Gly Gly Gly Lys 2025 226 26 PRT Artificial Sequence serum albumin-binding agent 226 GlyAsp Ser Pro Tyr Cys Lys Ile Ala Leu Phe Gln His Phe Glu Val 1 5 10 15Cys Ala Ala Asp Asp Pro Gly Gly Gly Lys 20 25 227 26 PRT ArtificialSequence serum albumin-binding agent 227 Gly Asp Pro Arg Ser Cys Val GluLys Tyr Tyr Trp Asp Val Leu Ile 1 5 10 15 Cys Gly Phe Phe Asp Pro GlyGly Gly Lys 20 25 228 24 PRT Artificial Sequence serum albumin-bindingagent 228 Gly Ser Arg Ser Phe Cys Met Asp Trp Pro Asn His Arg Asp CysAsp 1 5 10 15 Tyr Ser Ala Pro Gly Gly Gly Lys 20 229 22 PRT ArtificialSequence serum albumin-binding agent 229 Ala Gly Lys Trp Tyr Cys Ala TrpAsp Pro Leu Val Cys Glu Ile Phe 1 5 10 15 Gly Thr Gly Gly Gly Lys 20 23022 PRT Artificial Sequence serum albumin-binding agent 230 Ala Gly TrpThr Thr Cys His Ile Tyr Asp Trp Phe Cys Ser Ser Ser 1 5 10 15 Gly ThrGly Gly Gly Lys 20 231 22 PRT Artificial Sequence serum albumin-bindingagent 231 Ala Gly Leu Glu Tyr Cys Phe Tyr Gln Trp Trp Gly Cys Pro HisAla 1 5 10 15 Gly Thr Gly Gly Gly Lys 20 232 22 PRT Artificial Sequenceserum albumin-binding agent 232 Ala Gly Tyr Gln Phe Cys Thr Trp Asp ProIle Phe Cys Gly Trp His 1 5 10 15 Gly Thr Gly Gly Gly Lys 20 233 20 PRTArtificial Sequence serum albumin-binding agent 233 Gly Ser Leu Trp AspCys Trp Leu Tyr Asp Cys Glu Gly Asn Ala Pro 1 5 10 15 Gly Gly Gly Lys 20234 8 PRT Artificial Sequence exemplary motif 234 Xaa Xaa Xaa Xaa XaaTrp Cys Xaa 1 5 235 8 PRT Artificial Sequence exemplary motif 235 XaaXaa Trp Xaa Xaa Xaa Trp Xaa 1 5 236 6 PRT Artificial Sequence exemplarymotif 236 Xaa Trp Xaa Trp Trp Xaa 1 5 237 9 PRT Artificial Sequenceexemplary motif 237 Xaa Pro Xaa Trp Xaa Cys Xaa Xaa Xaa 1 5 238 18 PRTArtificial Sequence immunoglobulin binding polypeptide 238 Arg Arg AlaCys Ser Arg Asp Trp Ser Gly Ala Leu Val Trp Cys Ala 1 5 10 15 Gly His239 18 PRT Artificial Sequence immunoglobulin binding polypeptide 239Trp Gly Glu Cys Thr Val Thr Ser Tyr Gly Glu Leu Ile Trp Cys Gly 1 5 1015 Gly Leu 240 18 PRT Artificial Sequence immunoglobulin bindingpolypeptide 240 Ser Ser Ala Cys Ala Phe Asp Pro Met Gly Ala Val Ile TrpCys Thr 1 5 10 15 Tyr Asp 241 18 PRT Artificial Sequence immunoglobulinbinding polypeptide 241 Leu Leu Glu Cys Ala Tyr Asn Thr Ser Gly Glu LeuIle Trp Cys Asn 1 5 10 15 Gly Ser 242 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 242 Pro Asp Asp Cys Ser Ile His PheSer Gly Glu Leu Ile Trp Cys Glu 1 5 10 15 Pro Leu 243 18 PRT ArtificialSequence immunoglobulin binding polypeptide 243 Leu Gly Glu Cys Thr ValThr Ser Tyr Gly Glu Leu Ile Trp Cys Gly 1 5 10 15 Gly Leu 244 18 PRTArtificial Sequence immunoglobulin binding polypeptide 244 Trp Gly GluCys Thr Val Thr Ser Tyr Gly Glu Leu Ile Trp Cys Gly 1 5 10 15 Gly His245 18 PRT Artificial Sequence immunoglobulin binding polypeptide 245Asp His Met Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile Trp Cys Asp 1 5 1015 Asp His 246 18 PRT Artificial Sequence immunoglobulin bindingpolypeptide 246 Trp Gly Glu Cys Thr Val Thr Ser Tyr Gly Glu Leu Ile TrpCys Gly 1 5 10 15 Gly Leu 247 18 PRT Artificial Sequence immunoglobulinbinding polypeptide 247 Cys Arg Ala Cys Ser Arg Asp Trp Pro Gly Ala LeuVal Trp Cys Ala 1 5 10 15 Gly His 248 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 248 Arg Arg Ala Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala 1 5 10 15 Gly His 249 18 PRT ArtificialSequence immunoglobulin binding polypeptide 249 Leu His Ala Cys Ala PheAsp Pro Met Gly Ala Val Ile Trp Cys Thr 1 5 10 15 Tyr Asp 250 18 PRTArtificial Sequence immunoglobulin binding polypeptide 250 Asp His MetCys Val Tyr Thr Thr Trp Gly Glu Leu Met Trp Cys Asp 1 5 10 15 Asn His251 18 PRT Artificial Sequence immunoglobulin binding polypeptide 251Pro Pro Thr Cys Thr Trp Asp Trp Gln Gly Ile Leu Val Trp Cys Ser 1 5 1015 Gly His 252 18 PRT Artificial Sequence immunoglobulin bindingpolypeptide 252 Ser Asn Lys Cys Ser Asn Thr Trp Asp Gly Ser Leu Ile TrpCys Ser 1 5 10 15 Ala Asn 253 18 PRT Artificial Sequence immunoglobulinbinding polypeptide 253 Phe Pro Glu Cys Thr Phe Asp Met Glu Gly Phe LeuIle Trp Cys Ser 1 5 10 15 Ser Phe 254 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 254 His Asp Leu Cys Ala Gln Ala ProPhe Gly Asp Ala Thr Trp Cys Asp 1 5 10 15 Leu Arg 255 18 PRT ArtificialSequence immunoglobulin binding polypeptide 255 Pro Asn His Cys Ser TyrAsn Leu Lys Ser Glu Leu Ile Trp Cys Gln 1 5 10 15 Asp Leu 256 18 PRTArtificial Sequence immunoglobulin binding polypeptide 256 Pro Leu AspCys Ala Arg Asp Ile His Asn Ser Leu Ile Trp Cys Ser 1 5 10 15 Leu Gly257 18 PRT Artificial Sequence immunoglobulin binding polypeptide 257Gly Ser Glu Cys Ser Trp Thr Ser Leu Asn Glu Leu Ile Trp Cys Ala 1 5 1015 His Trp 258 18 PRT Artificial Sequence immunoglobulin bindingpolypeptide 258 Trp Pro Asp Cys Ser Phe Thr Val Gln Arg Asp Leu Ile TrpCys Glu 1 5 10 15 Ala Leu 259 18 PRT Artificial Sequence immunoglobulinbinding polypeptide 259 Ser His Ser Cys Ala Tyr Asp Tyr Ala His Met LeuVal Trp Cys Thr 1 5 10 15 His Phe 260 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 260 Asp His Met Cys Val Tyr Thr ThrTrp Gly Glu Leu Ile Trp Cys Asp 1 5 10 15 Asn His 261 18 PRT ArtificialSequence immunoglobulin binding polypeptide 261 Arg Pro Asn Cys Thr PheAla Ala Ser Gly Glu Leu Ile Trp Cys Met 1 5 10 15 His Tyr 262 18 PRTArtificial Sequence immunoglobulin binding polypeptide 262 Trp Trp GlyCys Gln Phe Asp Trp Arg Gly Glu Leu Val Trp Cys Pro 1 5 10 15 Tyr Leu263 18 PRT Artificial Sequence immunoglobulin binding polypeptide 263Gly Gly Val Cys Ser Tyr Ser Gly Met Gly Glu Ile Val Trp Cys Arg 1 5 1015 Trp Phe 264 18 PRT Artificial Sequence immunoglobulin bindingpolypeptide 264 Ala Leu Met Cys Ser His Asp Met Trp Gly Ser Leu Ile TrpCys Lys 1 5 10 15 His Phe 265 18 PRT Artificial Sequence immunoglobulinbinding polypeptide 265 Trp Trp Asn Cys His Asn Gly Trp Thr Trp Thr GlyGly Trp Cys Trp 1 5 10 15 Trp Phe 266 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 266 Tyr His Val Cys Ala Arg Asp SerTrp Asp Gln Leu Ile Trp Cys Glu 1 5 10 15 Ala Phe 267 15 PRT ArtificialSequence immunoglobulin binding polypeptide 267 Asn Tyr Trp Cys Asn PheTrp Gln Leu Pro Thr Cys Asp Asn Leu 1 5 10 15 268 16 PRT ArtificialSequence immunoglobulin binding polypeptide 268 Tyr Trp Tyr Cys Lys TrpPhe Ser Glu Ser Ala Ser Cys Ser Ser Arg 1 5 10 15 269 16 PRT ArtificialSequence immunoglobulin binding polypeptide 269 Tyr Trp Tyr Cys Lys TrpPhe Glu Asp Lys His Pro Cys Asp Ser Ser 1 5 10 15 270 16 PRT ArtificialSequence immunoglobulin binding polypeptide 270 Tyr Trp Tyr Cys Ser TrpPhe Pro Asp Arg Pro Asp Cys Pro Leu Tyr 1 5 10 15 271 16 PRT ArtificialSequence immunoglobulin binding polypeptide 271 Asn Tyr Trp Cys Asn ValTrp Leu Leu Gly Asp Val Cys Arg Ser His 1 5 10 15 272 18 PRT ArtificialSequence immunoglobulin binding polypeptide 272 Leu Tyr Trp Cys His ValTrp Phe Gly Gln His Ala Trp Gln Cys Lys 1 5 10 15 Tyr Pro 273 14 PRTArtificial Sequence immunoglobulin binding polypeptide 273 Tyr Trp LysCys Lys Trp Met Pro Trp Met Cys Gly Phe Asp 1 5 10 274 18 PRT ArtificialSequence immunoglobulin binding polypeptide 274 Asp Asp His Cys Tyr TrpPhe Arg Glu Trp Phe Asn Ser Glu Cys Pro 1 5 10 15 His Gly 275 15 PRTArtificial Sequence immunoglobulin binding polypeptide 275 Asn Tyr TrpCys Asn Ile Trp Gly Leu His Gly Cys Asn Ser His 1 5 10 15 276 16 PRTArtificial Sequence immunoglobulin binding polypeptide 276 Tyr Trp PheCys Gln Trp Phe Ser Gln Asn His Thr Cys Phe Arg Asp 1 5 10 15 277 16 PRTArtificial Sequence immunoglobulin binding polypeptide 277 His Tyr TrpCys Asp Ile Trp Phe Gly Ala Pro Ala Cys Gln Phe Arg 1 5 10 15 278 17 PRTArtificial Sequence immunoglobulin binding polypeptide 278 Ser Gly AspCys Gly Phe Trp Pro Arg Ile Trp Gly Leu Cys Met Asp 1 5 10 15 Asn 279 16PRT Artificial Sequence immunoglobulin binding polypeptide 279 Phe TrpTyr Cys Lys Trp Phe Tyr Glu Asp Ala Gln Cys Ser His Asp 1 5 10 15 280 13PRT Artificial Sequence immunoglobulin binding polypeptide 280 Tyr TyrTrp Cys Asn Tyr Trp Gly Leu Cys Pro Asp Gln 1 5 10 281 13 PRT ArtificialSequence immunoglobulin binding polypeptide 281 Ser Tyr Trp Cys Lys IleTrp Asp Val Cys Pro Gln Ser 1 5 10 282 13 PRT Artificial Sequenceimmunoglobulin binding polypeptide 282 Lys Tyr Trp Cys Asn Leu Trp GlyVal Cys Pro Ala Asn 1 5 10 283 13 PRT Artificial Sequence immunoglobulinbinding polypeptide 283 Gln Tyr Trp Cys Tyr Gln Trp Gly Leu Cys Gly AlaAsn 1 5 10 284 13 PRT Artificial Sequence immunoglobulin bindingpolypeptide 284 Lys Tyr Trp Cys Gln Gln Trp Gly Val Cys Asn Gly Ser 1 510 285 13 PRT Artificial Sequence immunoglobulin binding polypeptide 285Lys Tyr Trp Cys Val Gln Trp Gly Val Cys Pro Glu Ser 1 5 10 286 13 PRTArtificial Sequence immunoglobulin binding polypeptide 286 Lys Tyr TrpCys Met Gln Trp Gly Leu Cys Gly Trp Glu 1 5 10 287 13 PRT ArtificialSequence immunoglobulin binding polypeptide 287 His Phe Trp Cys Glu ValTrp Gly Leu Cys Pro Ser Ile 1 5 10 288 13 PRT Artificial Sequenceimmunoglobulin binding polypeptide 288 Gln Tyr Trp Cys Thr Lys Trp GlyLeu Cys Thr Asn Val 1 5 10 289 13 PRT Artificial Sequence immunoglobulinbinding polypeptide 289 Ala Tyr Trp Cys Lys Val Trp Gly Leu Cys Gln GlyGlu 1 5 10 290 13 PRT Artificial Sequence immunoglobulin bindingpolypeptide 290 Lys Tyr Trp Cys Asn Leu Trp Gly Val Cys Pro Ala Asn 1 510 291 13 PRT Artificial Sequence immunoglobulin binding polypeptide 291Gln Tyr Trp Cys Asn Val Trp Gly Val Cys Leu Pro Ser 1 5 10 292 13 PRTArtificial Sequence immunoglobulin binding polypeptide 292 His Tyr TrpCys Gln Gln Trp Gly Ile Cys Glu Arg Pro 1 5 10 293 13 PRT ArtificialSequence immunoglobulin binding polypeptide 293 Arg Tyr Trp Cys Asn IleTrp Asp Val Cys Pro Glu Gln 1 5 10 294 13 PRT Artificial Sequenceimmunoglobulin binding polypeptide 294 Gln Tyr Trp Cys Thr His Trp GlyLeu Cys Gly Lys Tyr 1 5 10 295 13 PRT Artificial Sequence immunoglobulinbinding polypeptide 295 Thr Tyr Trp Cys Thr Lys Trp Gly Leu Cys Pro HisAsn 1 5 10 296 13 PRT Artificial Sequence immunoglobulin bindingpolypeptide 296 Phe Tyr Trp Cys Gly Gln Trp Gly Leu Cys Ala Pro Pro 1 510 297 13 PRT Artificial Sequence immunoglobulin binding polypeptide 297Gly Tyr Trp Cys Asn Val Trp Gly Leu Cys Ser Thr Glu 1 5 10 298 13 PRTArtificial Sequence immunoglobulin binding polypeptide 298 Arg Tyr TrpCys Gly Val Trp Gly Val Cys Glu Ile Asp 1 5 10 299 13 PRT ArtificialSequence immunoglobulin binding polypeptide 299 Lys Phe Trp Cys Thr IleTrp Gly Val Cys His Met Pro 1 5 10 300 13 PRT Artificial Sequenceimmunoglobulin binding polypeptide 300 His Tyr Trp Cys Gln Gln Trp GlyIle Cys Glu Arg Pro 1 5 10 301 13 PRT Artificial Sequence immunoglobulinbinding polypeptide 301 Arg Tyr Trp Cys Asn Ile Trp Asp Val Cys Pro GluGln 1 5 10 302 13 PRT Artificial Sequence immunoglobulin bindingpolypeptide 302 Phe Tyr Trp Cys Ser Gln Trp Gly Leu Cys Lys Tyr Asp 1 510 303 13 PRT Artificial Sequence immunoglobulin binding polypeptide 303His Tyr Trp Cys Glu Lys Trp Gly Leu Cys Leu Met Ser 1 5 10 304 13 PRTArtificial Sequence immunoglobulin binding polypeptide 304 His Tyr TrpCys Gln Lys Trp Gly Val Cys Pro Thr Asp 1 5 10 305 13 PRT ArtificialSequence immunoglobulin binding polypeptide 305 His Tyr Trp Cys Ser LeuTrp Gly Val Cys Asp Ile Asn 1 5 10 306 12 PRT Artificial Sequenceimmunoglobulin binding polypeptide 306 Arg Phe Trp Cys Ser Ala Trp GlyVal Cys Pro Ala 1 5 10 307 13 PRT Artificial Sequence immunoglobulinbinding polypeptide 307 Ser Tyr Trp Cys Lys Ile Trp Asp Val Cys Pro GlnSer 1 5 10 308 13 PRT Artificial Sequence immunoglobulin bindingpolypeptide 308 Gln Tyr Trp Cys Ser Ile Trp Lys Val Cys Pro Gly Arg 1 510 309 13 PRT Artificial Sequence immunoglobulin binding polypeptide 309Tyr Trp Tyr Cys Glu Trp Phe Gly Ala Cys Ile Asn Asp 1 5 10 310 14 PRTArtificial Sequence immunoglobulin binding polypeptide 310 Glu Tyr TrpCys Lys Tyr Trp Gly Leu Glu Cys Val His Arg 1 5 10 311 14 PRT ArtificialSequence immunoglobulin binding polypeptide 311 Lys Tyr Trp Cys Thr GlnTrp Gly Leu Lys Cys Asp Lys Gln 1 5 10 312 13 PRT Artificial Sequenceimmunoglobulin binding polypeptide 312 Lys Tyr Trp Cys Ser Phe Trp GlyLeu Gln Cys Lys Thr 1 5 10 313 14 PRT Artificial Sequence immunoglobulinbinding polypeptide 313 Arg Tyr Trp Cys Asn Phe Trp Gly Val Asn Cys AspAla Asn 1 5 10 314 14 PRT Artificial Sequence immunoglobulin bindingpolypeptide 314 Asn Tyr Trp Cys Thr His Trp Gly Val Met Cys Leu Asp His1 5 10 315 14 PRT Artificial Sequence immunoglobulin binding polypeptide315 Tyr Trp Phe Cys Lys Trp Phe Pro Ser Gln Cys Gln Phe Met 1 5 10 31614 PRT Artificial Sequence immunoglobulin binding polypeptide 316 AlaTyr Trp Cys Lys Gln Trp Gly Leu Lys Cys Gln Leu Gly 1 5 10 317 14 PRTArtificial Sequence immunoglobulin binding polypeptide 317 Lys Tyr TrpCys Lys Phe Trp Gly Leu Glu Cys Lys Val Gly 1 5 10 318 14 PRT ArtificialSequence immunoglobulin binding polypeptide 318 Asn Tyr Trp Cys Thr GluTrp Gly Leu Asn Cys Asn Asn Lys 1 5 10 319 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 319 Ser Tyr Trp Cys Glu Lys Trp GlyLeu Thr Cys Glu Thr His 1 5 10 320 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 320 Glu Tyr Trp Cys Arg Ile Trp GlyLeu Gln Cys Asn Met Val 1 5 10 321 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 321 Lys Tyr Trp Cys Lys Lys Trp GlyVal Asn Cys Asp Phe Asn 1 5 10 322 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 322 Lys Tyr Trp Cys Ser Val Trp GlyVal Gln Cys Pro His Ser 1 5 10 323 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 323 Phe Tyr Trp Cys Thr Lys Trp GlyLeu Glu Cys Ile His Ser 1 5 10 324 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 324 His Tyr Trp Cys Gln Gln Trp GlyLeu Met Cys Phe Glu Thr 1 5 10 325 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 325 Lys Tyr Trp Cys Lys Arg Trp GlyLeu Met Cys Asn Gly Gly 1 5 10 326 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 326 Ala Tyr Trp Cys Met Thr Trp GlyVal Pro Cys Ile Ser Trp 1 5 10 327 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 327 Lys Tyr Trp Cys Lys Lys Trp GlyVal Asn Cys Asp Phe Asn 1 5 10 328 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 328 Lys Tyr Trp Cys Ser Val Trp GlyVal Gln Cys Pro Asp Ser 1 5 10 329 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 329 Lys Tyr Trp Cys Ser Val Trp GlyVal Gln Cys Pro His Ser 1 5 10 330 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 330 Leu Tyr Trp Cys Thr Lys Trp GlyVal Thr Cys Gln Lys Asp 1 5 10 331 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 331 Thr Tyr Trp Cys His Lys Trp GlyVal Lys Cys Ala Thr Thr 1 5 10 332 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 332 Thr Tyr Trp Cys Thr Phe Trp GluLeu Pro Cys Asp Pro Ala 1 5 10 333 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 333 Lys Tyr Trp Cys Thr Lys Trp GlnLeu Asn Cys Glu Glu Val 1 5 10 334 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 334 Asn Tyr Trp Cys His Phe Trp GlnVal Pro Cys Leu Glu Gln 1 5 10 335 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 335 Thr Tyr Trp Cys Val Val Trp AsnVal Pro Cys Ser Thr Asp 1 5 10 336 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 336 Asn Phe Trp Cys His Thr Trp GlyLeu Gln Cys Asn Asp Leu 1 5 10 337 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 337 Phe Trp Tyr Cys Tyr Trp Phe AsnGlu Lys Cys Lys Thr Pro 1 5 10 338 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 338 Gly Phe Trp Cys Thr Phe Trp GlyVal Thr Cys Glu Ala Gly 1 5 10 339 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 339 Pro His Asn Cys Asp Asp His TyrTrp Tyr Cys Lys Trp Phe 1 5 10 340 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 340 Glu Met Thr Cys Ser Ser His TyrTrp Tyr Cys Thr Trp Met 1 5 10 341 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 341 His Ile Asp Cys Lys Thr Asn TyrTrp Trp Cys Arg Trp Thr 1 5 10 342 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 342 Glu Met Arg Cys Gly Gln His PheTrp Tyr Cys Glu Trp Phe 1 5 10 343 15 PRT Artificial Sequenceimmunoglobulin binding polypeptide 343 Asn Tyr Trp Cys Asn Phe Trp GlnLeu Pro Thr Cys Asp Asn Leu 1 5 10 15 344 16 PRT Artificial Sequenceimmunoglobulin binding polypeptide 344 Tyr Trp Tyr Cys Gln Trp Phe GlnGlu Val Asn Lys Cys Phe Asn Ser 1 5 10 15 345 16 PRT Artificial Sequenceimmunoglobulin binding polypeptide 345 Tyr Tyr Trp Cys Arg His Trp PhePro Asp Phe Asp Cys Val His Ser 1 5 10 15 346 16 PRT Artificial Sequenceimmunoglobulin binding polypeptide 346 Tyr Trp Tyr Cys Ser Trp Phe ProAsp Arg Pro Asp Cys Pro Leu Tyr 1 5 10 15 347 16 PRT Artificial Sequenceimmunoglobulin binding polypeptide 347 Tyr Trp Tyr Cys Val Trp Phe AspAsn Ala Asp Gln Cys Val His His 1 5 10 15 348 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 348 Ala Ala Thr Cys Ser Thr Ser TyrTrp Tyr Tyr Gln Trp Phe Cys Thr 1 5 10 15 Asp Ser 349 14 PRT ArtificialSequence immunoglobulin binding polypeptide 349 Tyr Trp Ala Cys Val TrpGly Leu Lys Ser Cys Val Asp Arg 1 5 10 350 13 PRT Artificial Sequenceimmunoglobulin binding polypeptide 350 Tyr Trp Arg Cys Val Trp Phe ProAla Ser Cys Pro Thr 1 5 10 351 18 PRT Artificial Sequence immunoglobulinbinding polypeptide 351 Asp Trp Gln Cys Leu Trp Trp Gly Asn Ser Phe TrpPro Tyr Cys Ala 1 5 10 15 Asn Leu 352 14 PRT Artificial Sequenceimmunoglobulin binding polypeptide 352 Phe Trp Arg Cys His Trp Trp ProGlu Arg Cys Pro Val Asp 1 5 10 353 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 353 Asn Pro Met Cys Trp Lys Lys SerTrp Trp Glu Asp Ala Tyr Cys Ile 1 5 10 15 Asn His 354 18 PRT ArtificialSequence immunoglobulin binding polypeptide 354 Ser Trp Val Cys Trp LysAla Lys Trp Trp Glu Asp Lys Arg Cys Ala 1 5 10 15 Pro Phe 355 18 PRTArtificial Sequence immunoglobulin binding polypeptide 355 Ser Arg GlnCys Trp Lys Glu Leu Trp Trp Thr Asp Gln Met Cys Leu 1 5 10 15 Asp Leu356 16 PRT Artificial Sequence immunoglobulin binding polypeptide 356Ser Phe Arg Cys Gln Ser Ser Phe Pro Ser Trp Tyr Cys Asp Tyr Tyr 1 5 1015 357 16 PRT Artificial Sequence immunoglobulin binding polypeptide 357Ser Trp His Cys Gln Asn Thr Tyr Pro Glu Trp Tyr Cys Gln Trp Tyr 1 5 1015 358 17 PRT Artificial Sequence immunoglobulin binding polypeptide 358Gly Ser Lys Cys Lys Gln Thr Gly Phe Pro Arg Trp Trp Cys Glu His 1 5 1015 Tyr 359 18 PRT Artificial Sequence immunoglobulin binding polypeptide359 Asp Gly Val Cys Gly Pro Arg Gly Phe Gly Pro Ala Trp Phe Cys Met 1 510 15 His Tyr 360 16 PRT Artificial Sequence immunoglobulin bindingpolypeptide 360 Tyr Ser His Cys Ala Thr His Tyr Pro Thr Trp Tyr Cys LeuHis Phe 1 5 10 15 361 16 PRT Artificial Sequence immunoglobulin bindingpolypeptide 361 Phe Cys Asn Cys Trp Gly Ser His Glu Phe Thr Phe Cys ValAsp Asp 1 5 10 15 362 18 PRT Artificial Sequence immunoglobulin bindingpolypeptide 362 Pro Gly Trp Cys Tyr Ser Asp Ile Trp Gly Phe Lys His PheCys Asn 1 5 10 15 Leu Asp 363 16 PRT Artificial Sequence immunoglobulinbinding polypeptide 363 Asp Ser Ser Cys Ile Lys His His Asn Lys Val ThrCys Phe Phe Pro 1 5 10 15 364 13 PRT Artificial Sequence immunoglobulinbinding polypeptide 364 Arg Trp Ser Cys Trp Gly Val Trp Gly Cys Val TrpVal 1 5 10 365 14 PRT Artificial Sequence immunoglobulin bindingpolypeptide 365 Pro Val Asp Cys Lys His His Phe Trp Trp Cys Tyr Trp Asn1 5 10 366 17 PRT Artificial Sequence immunoglobulin binding polypeptide366 Ser Trp Asn Cys Ala Phe His His Asn Glu Met Val Trp Cys Asp Asp 1 510 15 Gly 367 15 PRT Artificial Sequence immunoglobulin bindingpolypeptide 367 Tyr Trp Tyr Cys Trp Phe Pro Asp Arg Pro Glu Cys Pro LeuTyr 1 5 10 15 368 29 PRT Artificial Sequence immunoglobulin bindingpolypeptide 368 Gly Asp Asp His Met Cys Val Tyr Thr Thr Trp Gly Glu LeuIle Trp 1 5 10 15 Cys Asp Asn His Glu Pro Gly Pro Glu Gly Gly Gly Lys 2025 369 29 PRT Artificial Sequence immunoglobulin binding polypeptide 369Gly Asp Arg Arg Ala Cys Ser Arg Asp Trp Ser Gly Ala Leu Val Trp 1 5 1015 Cys Ala Gly His Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 370 25 PRTArtificial Sequence immunoglobulin binding polypeptide 370 Ala Gly LysTyr Trp Cys Ser Phe Trp Gly Leu Gln Cys Lys Thr Gly 1 5 10 15 Thr ProGly Pro Glu Gly Gly Gly Lys 20 25 371 23 PRT Artificial Sequenceimmunoglobulin binding polypeptide 371 Ala Gly Ser Tyr Trp Cys Lys IleTrp Asp Val Cys Pro Gln Ser Pro 1 5 10 15 Gly Pro Glu Gly Gly Gly Lys 20372 23 PRT Artificial Sequence immunoglobulin binding polypeptide 372Ala Gly Lys Tyr Trp Cys Asn Leu Trp Gly Val Cys Pro Ala Asn Pro 1 5 1015 Gly Pro Glu Gly Gly Gly Lys 20 373 24 PRT Artificial Sequenceimmunoglobulin binding polypeptide 373 Ala Gly Thr Tyr Trp Cys Thr PheTrp Glu Leu Pro Cys Asp Pro Ala 1 5 10 15 Pro Gly Pro Glu Gly Gly GlyLys 20 374 24 PRT Artificial Sequence immunoglobulin binding polypeptide374 Ala Gly Pro His Asn Cys Asp Asp His Tyr Trp Tyr Cys Lys Trp Phe 1 510 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 375 28 PRT Artificial Sequenceimmunoglobulin binding polypeptide 375 Ala Gly Ala Ala Thr Cys Ser ThrSer Tyr Trp Tyr Tyr Gln Trp Phe 1 5 10 15 Cys Thr Asp Ser Pro Gly ProGlu Gly Gly Gly Lys 20 25 376 25 PRT Artificial Sequence immunoglobulinbinding polypeptide 376 Ala Gly Tyr Trp Tyr Cys Trp Phe Pro Asp Arg ProGlu Cys Pro Leu 1 5 10 15 Tyr Pro Gly Pro Glu Gly Gly Gly Lys 20 25 37726 PRT Artificial Sequence immunoglobulin binding polypeptide 377 AlaGly Pro Val Asp Cys Lys His His Phe Trp Trp Cys Tyr Trp Asn 1 5 10 15Gly Thr Pro Gly Pro Glu Gly Gly Gly Lys 20 25 378 29 PRT ArtificialSequence immunoglobulin binding polypeptide 378 Gly Asp Asp Asp His CysTyr Trp Phe Arg Glu Trp Phe Asn Ser Glu 1 5 10 15 Cys Pro His Gly GluPro Gly Pro Glu Gly Gly Gly Lys 20 25 379 25 PRT Artificial Sequenceimmunoglobulin binding polypeptide 379 Ala Gly Tyr Tyr Trp Cys Asn TyrTrp Gly Leu Cys Pro Asp Gln Gly 1 5 10 15 Thr Pro Gly Pro Glu Gly GlyGly Lys 20 25 380 30 PRT Artificial Sequence immunoglobulin bindingpolypeptide 380 Gly Asp Ser Trp Val Cys Trp Lys Ala Lys Trp Trp Glu AspLys Arg 1 5 10 15 Cys Ala Pro Phe Gly Thr Pro Gly Pro Glu Gly Gly GlyLys 20 25 30 381 30 PRT Artificial Sequence immunoglobulin bindingpolypeptide 381 Gly Asp Asn Pro Met Cys Trp Lys Lys Ser Trp Trp Glu AspAla Tyr 1 5 10 15 Cys Ile Asn His Gly Thr Pro Gly Pro Glu Gly Gly GlyLys 20 25 30 382 29 PRT Artificial Sequence immunoglobulin bindingpolypeptide 382 Gly Asp Ser Trp Asn Cys Ala Phe His His Asn Glu Met ValTrp Cys 1 5 10 15 Asp Asp Gly Gly Thr Pro Gly Pro Glu Gly Gly Gly Lys 2025 383 29 PRT Artificial Sequence immunoglobulin binding polypeptide 383Gly Asp Trp Gly Glu Cys Thr Val Thr Ser Tyr Gly Glu Leu Ile Trp 1 5 1015 Cys Gly Gly Leu Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 384 29 PRTArtificial Sequence immunoglobulin binding polypeptide 384 Gly Asp AsnPro Met Cys Trp Arg Ala Ser Trp Trp Glu Asp Ala Tyr 1 5 10 15 Cys IleAsn His Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 385 29 PRT ArtificialSequence immunoglobulin binding polypeptide 385 Gly Asp Asn Pro Met CysTrp Arg Ala His Trp Trp Glu Asp Ala Tyr 1 5 10 15 Cys Ile Asn His GluPro Gly Pro Glu Gly Gly Gly Lys 20 25 386 27 PRT Artificial Sequenceimmunoglobulin binding polypeptide 386 Gly Asp Asp His Met Cys Val TyrThr Thr Trp Gly Glu Leu Ile Trp 1 5 10 15 Cys Asp Asn His Glu Pro GlyPro Glu Gly Xaa 20 25 387 24 PRT Artificial Sequence immunoglobulinbinding polypeptide 387 Gly Asp Asp His Met Cys Val Tyr Thr Thr Trp GlyGlu Leu Ile Trp 1 5 10 15 Cys Asp Asn His Glu Pro Gly Xaa 20 388 24 PRTArtificial Sequence immunoglobulin binding polypeptide 388 Gly Asp AspHis Met Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile Trp 1 5 10 15 Cys AspAsn His Glu Pro Gly Xaa 20 389 21 PRT Artificial Sequence immunoglobulinbinding polypeptide 389 Gly Asp Asp His Met Cys Val Tyr Thr Thr Trp GlyGlu Leu Ile Trp 1 5 10 15 Cys Asp Asn His Xaa 20 390 21 PRT ArtificialSequence immunoglobulin binding polypeptide 390 Gly Asp Asp His Met CysVal Tyr Thr Thr Trp Gly Glu Leu Ile Trp 1 5 10 15 Cys Asp Asn His Xaa 20391 21 PRT Artificial Sequence immunoglobulin binding polypeptide 391Gly Asp Asp His Met Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile Trp 1 5 1015 Cys Asp Asn His Xaa 20 392 27 PRT Artificial Sequence immunoglobulinbinding polypeptide 392 Asp His Met Cys Val Tyr Thr Thr Trp Gly Glu LeuIle Trp Cys Asp 1 5 10 15 Asn His Glu Pro Gly Pro Glu Gly Gly Gly Lys 2025 393 27 PRT Artificial Sequence immunoglobulin binding polypeptide 393Glu His Met Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile Trp Cys Asp 1 5 1015 Asn His Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 394 25 PRTArtificial Sequence immunoglobulin binding polypeptide 394 Ala Cys ValTyr Thr Thr Trp Gly Glu Leu Ile Trp Cys Asp Asn His 1 5 10 15 Glu ProGly Pro Glu Gly Gly Gly Lys 20 25 395 25 PRT Artificial Sequenceimmunoglobulin binding polypeptide 395 Thr Cys Val Tyr Thr Thr Trp GlyGlu Leu Ile Trp Cys Asp Asn His 1 5 10 15 Glu Pro Gly Pro Glu Gly GlyGly Lys 20 25 396 25 PRT Artificial Sequence immunoglobulin bindingpolypeptide 396 Glu Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile Trp Cys AspAsn His 1 5 10 15 Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 397 25 PRTArtificial Sequence immunoglobulin binding polypeptide 397 Val Cys ValTyr Thr Thr Trp Gly Glu Leu Ile Trp Cys Asp Asn His 1 5 10 15 Glu ProGly Pro Glu Gly Gly Gly Lys 20 25 398 25 PRT Artificial Sequenceimmunoglobulin binding polypeptide 398 Xaa Cys Val Tyr Thr Thr Trp GlyGlu Leu Ile Trp Cys Asp Asn His 1 5 10 15 Glu Pro Gly Pro Glu Gly GlyGly Lys 20 25 399 24 PRT Artificial Sequence immunoglobulin bindingpolypeptide 399 Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile Trp Cys Asp AsnHis Glu 1 5 10 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 400 27 PRTArtificial Sequence immunoglobulin binding polypeptide 400 Ser Arg AlaCys Ser Arg Asp Trp Ser Gly Ala Leu Val Trp Cys Ala 1 5 10 15 Gly HisGlu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 401 27 PRT Artificial Sequenceimmunoglobulin binding polypeptide 401 Arg Arg Ala Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala 1 5 10 15 Gly His Glu Pro Gly Pro GluGly Gly Gly Lys 20 25 402 27 PRT Artificial Sequence immunoglobulinbinding polypeptide 402 Glu Arg Ala Cys Ser Arg Asp Trp Ser Gly Ala LeuVal Trp Cys Ala 1 5 10 15 Gly His Glu Pro Gly Pro Glu Gly Gly Gly Lys 2025 403 25 PRT Artificial Sequence immunoglobulin binding polypeptide 403Ala Cys Ser Arg Asp Trp Ser Gly Ala Leu Val Trp Cys Ala Gly His 1 5 1015 Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 404 25 PRT ArtificialSequence immunoglobulin binding polypeptide 404 Thr Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala Gly His 1 5 10 15 Glu Pro Gly Pro GluGly Gly Gly Lys 20 25 405 25 PRT Artificial Sequence immunoglobulinbinding polypeptide 405 Glu Cys Ser Arg Asp Trp Ser Gly Ala Leu Val TrpCys Ala Gly His 1 5 10 15 Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 40625 PRT Artificial Sequence immunoglobulin binding polypeptide 406 ValCys Ser Arg Asp Trp Ser Gly Ala Leu Val Trp Cys Ala Gly His 1 5 10 15Glu Pro Gly Pro Glu Gly Gly Gly Lys 20 25 407 25 PRT Artificial Sequenceimmunoglobulin binding polypeptide 407 Gly Cys Ser Arg Asp Trp Ser GlyAla Leu Val Trp Cys Ala Gly His 1 5 10 15 Glu Pro Gly Pro Glu Gly GlyGly Lys 20 25 408 24 PRT Artificial Sequence immunoglobulin bindingpolypeptide 408 Cys Ser Arg Asp Trp Ser Gly Ala Leu Val Trp Cys Ala GlyHis Glu 1 5 10 15 Pro Gly Pro Glu Gly Gly Gly Lys 20 409 18 PRTArtificial Sequence immunoglobulin binding polypeptide 409 Asn Pro MetCys Trp Arg Ala Ser Trp Trp Glu Asp Ala Tyr Cys Ile 1 5 10 15 Asn His410 18 PRT Artificial Sequence immunoglobulin binding polypeptide 410Asn Pro Met Cys Trp Arg Ala His Trp Trp Glu Asp Ala Tyr Cys Ile 1 5 1015 Asn His 411 18 PRT Artificial Sequence immunoglobulin bindingpolypeptide 411 Glu His Met Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile TrpCys Asp 1 5 10 15 Asn His 412 16 PRT Artificial Sequence immunoglobulinbinding polypeptide 412 Ala Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile TrpCys Asp Asn His 1 5 10 15 413 16 PRT Artificial Sequence immunoglobulinbinding polypeptide 413 Thr Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile TrpCys Asp Asn His 1 5 10 15 414 16 PRT Artificial Sequence immunoglobulinbinding polypeptide 414 Glu Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile TrpCys Asp Asn His 1 5 10 15 415 16 PRT Artificial Sequence immunoglobulinbinding polypeptide 415 Val Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile TrpCys Asp Asn His 1 5 10 15 416 16 PRT Artificial Sequence immunoglobulinbinding polypeptide 416 Xaa Cys Val Tyr Thr Thr Trp Gly Glu Leu Ile TrpCys Asp Asn His 1 5 10 15 417 18 PRT Artificial Sequence immunoglobulinbinding polypeptide 417 Ser Arg Ala Cys Ser Arg Asp Trp Ser Gly Ala LeuVal Trp Cys Ala 1 5 10 15 Gly His 418 18 PRT Artificial Sequenceimmunoglobulin binding polypeptide 418 Glu Arg Ala Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala 1 5 10 15 Gly His 419 16 PRT ArtificialSequence immunoglobulin binding polypeptide 419 Ala Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala Gly His 1 5 10 15 420 16 PRT ArtificialSequence immunoglobulin binding polypeptide 420 Thr Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala Gly His 1 5 10 15 421 16 PRT ArtificialSequence immunoglobulin binding polypeptide 421 Glu Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala Gly His 1 5 10 15 422 16 PRT ArtificialSequence immunoglobulin binding polypeptide 422 Val Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala Gly His 1 5 10 15 423 16 PRT ArtificialSequence immunoglobulin binding polypeptide 423 Gly Cys Ser Arg Asp TrpSer Gly Ala Leu Val Trp Cys Ala Gly His 1 5 10 15 424 15 PRT ArtificialSequence template sequence 424 Xaa Xaa Xaa Cys Xaa Xaa Xaa Xaa Xaa XaaXaa Cys Xaa Xaa Xaa 1 5 10 15 425 18 PRT Artificial Sequence Exemplarymotif 425 Xaa Arg Xaa Cys Xaa Thr Xaa Xaa Pro Xaa Xaa Xaa Xaa Xaa CysXaa 1 5 10 15 Xaa Xaa 426 18 PRT Artificial Sequence examplary motif 426Xaa Xaa Xaa Cys Ile Thr Xaa Pro Phe Xaa Xaa Xaa Xaa Xaa Cys Xaa 1 5 1015 Asn Xaa 427 12 PRT Artificial Sequence immunoglobulin segment 427 CysVal Tyr Thr Thr Trp Gly Glu Leu Ile Trp Cys 1 5 10 428 12 PRT ArtificialSequence immunoglobulin segment 428 Cys Ser Arg Asp Trp Ser Gly Ala LeuVal Trp Cys 1 5 10 429 12 PRT Artificial Sequence immunoglobulin segment429 Cys Ser Thr Ser Tyr Trp Tyr Tyr Gln Trp Phe Cys 1 5 10 430 18 PRTArtificial Sequence serum albumin - binding agent 430 Arg Asn Met CysLys Phe Ser Trp Ile Arg Ser Pro Ala Phe Cys Ala 1 5 10 15 Arg Ala

What is claimed:
 1. A method of evaluating a sample, the method comprising: providing a sample that comprises (i) a serum albumin, (ii) one or more compounds physically associated with the serum albumin and (iii) a serum albumin-binding agent that is free of an antigen-binding immunoglobulin variable domain; allowing the serum albumin-binding agent to bind to the serum albumin to form a complex; separating the complex from one or more components of the sample; and evaluating one or more of the physically associated compounds.
 2. A method of evaluating a sample, the method comprising: providing a sample that comprises (i) a serum albumin, (ii) one or more compounds physically associated with the serum albumin and (iii) a serum albumin-binding agent that comprises a peptide that independently binds to serum albumin; allowing the serum albumin-binding agent to bind to the serum albumin to form a complex; separating the complex from one or more components of the sample; and evaluating one or more of the physically associated compounds.
 3. The method of claim 1 or 2 wherein the serum albumin-binding agent binds serum albumin with an affinity of less than 5 μM.
 4. The method of claim 2 wherein the peptide is less than 30 amino acids in length.
 5. The method of claim 2 wherein the peptide comprises an intra-molecular disulfide bond.
 6. The method of claim 4 wherein the peptide comprises DX-236 or DX-321 or an amino acid sequence that differs from DX-236 or DX-321 by fewer than four amino acid substitutions.
 7. The method of claim 1 wherein the serum albumin-binding agent is coupled to an insoluble support.
 8. The method of claim 1 wherein the serum albumin-binding agent binds to serum albumin from a plurality of species.
 9. The method of claim 1 wherein at least one of the evaluated physically associated compounds is non-covalently associated with the serum albumin.
 10. The method of claim 9 further comprising separating one or more of the physically associated compounds from the serum albumin.
 11. The method of claim 10 wherein the separating of one or more of the physically associated compounds from the serum albumin is prior to the evaluating.
 12. The method of claim 1 or 2 further comprising separating the at least one non-covalently associated compounds from the serum albumin prior to the evaluating.
 13. The method of claim 12 wherein the separating from the serum albumin comprises covalently attaching the serum albumin to an insoluble support.
 14. The method of claim 13 wherein the covalent attachment is to a free cysteine of the serum albumin.
 15. A method of evaluating a sample, the method comprising: providing a sample that comprises (i) a serum albumin, (ii) one or more compounds physically associated with the serum albumin and (iii) a serum albumin-binding agent; allowing the serum albumin-binding agent to bind to the serum albumin to form a complex; separating the complex from one or more components of the sample; covalently attaching the serum albumin to an insoluble matrix; and separating at least one of the one or more compounds physically associated with the serum albumin from the serum albumin.
 16. The method of claim 15 wherein the covalent attachment is to a free cysteine of the serum albumin.
 17. The method of claim 15 further comprising evaluating one or more of the physically associated compounds that becomes separated from the serum albumin.
 18. The method of claim 15 wherein the covalent attachment is formed using a thiol reactive group.
 19. The method of claim 18 wherein the thiol reactive group comprises a halogen derivative.
 20. The method of claim 19 wherein the thiol reactive group comprises iodoacetamide.
 21. The method of claim 18 wherein the thiol reactive group comprises a maleimide.
 22. The method of claim 18 wherein the thiol reactive group comprises a thiol exchange reagent.
 23. The method of claim 22 wherein the thiol exchange reagent is a pyridyl disulfide.
 24. The method of claim 15 wherein the separating comprises denaturing the serum albumin.
 25. The method of claim 1 wherein at least one of the evaluated covalently associated compounds is non-proteinaceous.
 26. The method of claim 1 wherein the evaluating comprises one or more of: gel electrophoresis, mass spectroscopy, chromatography, and protein sequencing.
 27. The method of claim 1 wherein the evaluating comprises detecting a given compound using an affinity reagent specific for the given compound.
 28. The method of claim 27 wherein the affinity reagent is an antibody.
 29. The method of claim 1 wherein the evaluating comprises detecting a compound other than a fatty acid, hematin, and bilirubin.
 30. The method of claim 1 wherein the evaluating comprises detecting a polypeptide.
 31. The method of claim 1 wherein the evaluating comprises eluting an associated compound from the serum albumin by contacting the complex with a synthetic affinity ligand specific for an epitope on the serum albumin.
 32. The method of claim 1 wherein the evaluating comprises eluting an associated compound by contacting the complex with a natural compound that binds to the serum albumin.
 33. The method of claim 32 wherein the natural compound comprises a component selected from the group consisting of: a fatty acid, hematin, and bilirubin.
 34. The method of claim 32 wherein the natural compound comprises a negatively charged aromatic group having a molecular weight of less than 500 Daltons.
 35. The method of claim 1 wherein the serum albumin is a human serum albumin.
 36. The method of claim 1 wherein the serum albumin is an artificial mutant of a naturally-occurring serum albumin.
 37. The method of claim 1 further comprising digitally recording information that (i) indicates the presences or absence of a given compound among the evaluated one or more physically associated compounds, or (ii) describes the one or more physically associated compounds.
 38. The method of claim 1 further comprising providing a second sample, and evaluating one or more of the physically associated compounds in the second sample.
 39. The method of claim 38 further comprising comparing the results of evaluating the one or more of the physically associated compounds for the first sample to the second sample.
 40. The method of claim 39 wherein the first sample is from a first subject, and the second sample is from a second subject.
 41. The method of claim 40 wherein the first subject is treated with an agent, and the second subject is not treated with the agent.
 42. The method of claim 40 wherein the first subject and second subject are subjected to different environmental conditions.
 43. The method of claim 1 or 2 wherein the serum albumin-binding agent and the serum albumin preferentially dissociate in solutions above pH
 8. 44. The method of claim 1 wherein the sample is obtained from a subject.
 45. The method of claim 44 wherein the subject is a human.
 46. The method of claim 45 wherein the sample comprises blood or serum.
 47. The method of claim 45 wherein the sample is obtained from a biopsy.
 48. The method of claim 45 wherein the sample is obtained from a tumor or a region within 5 mm of a tumor.
 49. The method of claim 45 wherein the subject is treated with a therapeutic composition prior to obtaining the sample.
 50. The method of claim 49 wherein one or more of the evaluated physically associated compounds is an endogenous compound.
 51. The method of claim 49 wherein one or more of the evaluated physically associated compounds is a component of the therapeutic composition.
 52. A method of evaluating a sample, the method comprising: providing a sample that comprises (i) a soluble immunoglobulin protein that includes at least one immunoglobulin domain, (ii) one or more compounds physically associated with the immunoglobulin protein and (iii) immunoglobulin-binding agent that comprises a peptide that specifically binds to the immunoglobulin protein at a site other than an antigen binding site; allowing the immunoglobulin-binding agent to bind to the soluble immunoglobulin protein to form a complex that includes one or more compounds physically associated with the soluble immunoglobulin protein; separating the complex from one or more components of the sample; and evaluating one or more of the physically associated compounds.
 53. The method of claim 52 wherein the soluble immunoglobulin protein is a naturally-occurring protein.
 54. The method of claim 52 wherein the soluble immunoglobulin protein is an IgG.
 55. The method of claim 52 wherein the one or more physically associated compounds comprises an antigen.
 56. The method of claim 52 wherein the sample is obtained from a subject having an infection.
 57. The method of claim 52 wherein the sample is obtained from a subject having immunological disorder.
 58. The method of claim 57 wherein the immunological disorder is an auto-immune disorder.
 59. The method of claim 52 wherein the peptide is less than 30 amino acids in length.
 60. The method of claim 52 wherein the peptide comprises an intra-molecular disulfide bond.
 61. A method of evaluating a sample, the method comprising: providing a sample that comprises (i) a soluble serum protein, (ii) one or more compounds physically associated with the soluble serum protein, and (iii) serum protein-binding agent that comprises a peptide that specifically binds to the serum protein; allowing the serum protein-binding agent to bind to the soluble serum protein to form a complex that includes one or more compounds physically associated with the soluble serum protein; separating the complex from one or more components of the sample; and evaluating one or more of the physically associated compounds.
 62. The method of claim 61 wherein the serum protein is serum albumin.
 63. The method of claim 61 wherein the serum protein is at least 0.01% of the protein fraction in blood serum.
 64. The method of claim 61 wherein the serum protein is selected from the group consisting of: transferrin, a macroglobulins, ferritin, apolipoproteins, transthyretin, a protease inhibitor found in serum, retinol binding protein, thiostatin, a-fetoprotein, vitamin-D binding protein, or afamin.
 65. A method of mapping a physical interaction between serum albumin and an associated compound, the method comprising: providing a complex comprising a serum albumin and an associated compound; evaluating binding of a ligand (e.g., a peptide ligand described herein) to the complex, wherein the ligand binds to serum albumin with an affinity of less than 5 μM, the ligand is free of an immunoglobulin variable domain, and binding of the non-antibody ligand to the complex indicates that the associated compound does not bind an epitope that overlaps the epitope bound by the non-antibody ligand.
 66. The method of claim 65 further comprising: evaluating binding of a ligand to the complex, wherein the second ligand binds to serum albumin with an affinity of less than 5 μM.
 67. The method of claim 65 wherein one of the first and second non-antibody ligand binds is prevented from binding to the complex. 